Oil comprising polyunsaturated fatty acids

ABSTRACT

The present invention relates to a process for producing ethyl esters of polyunsaturated fatty acids, comprising transesterifying triacylglycerols in extracted plant lipid.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/678,008, filed Aug. 15, 2017, which is a continuation of U.S.application Ser. No. 15/400,532, filed Jan. 6, 2017, now U.S. Pat. No.9,932,289, issued Apr. 3, 2018, which is a continuation of U.S.application Ser. No. 14/600,653, filed Jan. 20, 2015, now U.S. Pat. No.9,556,102, issued Jan. 31, 2017, which is a continuation of U.S.application Ser. No. 13/918,392, filed Jun. 14, 2013, no U.S. Pat. No.8,946,460, issued Feb. 3, 2015, claiming the benefit of U.S. ProvisionalPatent Applications Nos. 61/782,680, filed Mar. 14, 2013, 61/697,676,filed Sep. 6, 2012, 61/663,344, filed Jun. 22, 2012, and 61/660,392,filed Jun. 15, 2012, the entire contents of each of which are herebyincorporated by reference into the subject application.

REFERENCE TO SEQUENCE LISTING

This application incorporates-by-reference nucleotide and/or amino acidsequences which are present in the file named“180424_84199-BAABA_Sequence_Listing_CAE.txt,” which is 371 kilobytes insize, and which was created Apr. 24, 2018 in the IBM-PC machine format,having an operating system compatibility with MS-Windows, which iscontained in the text file filed Apr. 24, 2018 as part of thisapplication.

FIELD OF THE INVENTION

The present invention relates to a process for producing ethyl esters ofpolyunsaturated fatty acids, comprising transesterifyingtriacylglycerols in extracted plant lipid.

BACKGROUND OF THE INVENTION

Omega-3 long-chain polyunsaturated fatty acids (LC-PUFA) are now widelyrecognized as important compounds for human and animal health. Thesefatty acids may be obtained from dietary sources or by conversion oflinoleic (LA, 18:2ω6) or α-linolenic (ALA, 18:3ω3) fatty acids, both ofwhich are regarded as essential fatty acids in the human diet. Whilehumans and many other vertebrate animals are able to convert LA or ALA,obtained from plant sources to C22 they carry out this conversion at avery low rate. Moreover, most modern societies have imbalanced diets inwhich at least 90% of polyunsaturated fatty acids (PUFA) are of the ω6fatty acids, instead of the 4:1 ratio or less for ω6:ω3 fatty acids thatis regarded as ideal (Trautwein, 2001). The immediate dietary source ofLC-PUFAs such as eicosapentaenoic acid (EPA, 20:5ω3) and docosahexaenoicacid (DHA, 22:6ω3) for humans is mostly from fish or fish oil. Healthprofessionals have therefore recommended the regular inclusion of fishcontaining significant levels of LC-PUFA into the human diet.Increasingly, fish-derived LC-PUFA oils are being incorporated into foodproducts and in infant formula, for example. However, due to a declinein global and national fisheries, alternative sources of thesebeneficial health-enhancing oils are needed.

Flowering plants, in contrast to animals, lack the capacity tosynthesise polyunsaturated fatty acids with chain lengths longer than 18carbons. In particular, crop and horticultural plants along with otherangiosperms do not have the enzymes needed to synthesize the longerchain ω3 fatty acids such as EPA, docosapentaenoic acid (DPA, 22:5ω3)and DHA that are derived from ALA. An important goal in plantbiotechnology is therefore the engineering of crop plants which producesubstantial quantities of LC-PUFA, thus providing an alternative sourceof these compounds.

LC-PUFA Biosynthesis Pathways

Biosynthesis of LC-PUFAs in organisms such as microalgae, mosses andfungi usually occurs as a series of oxygen-dependent desaturation andelongation reactions (FIG. 1). The most common pathway that produces EPAin these organisms includes a Δ6-desaturation, Δ6-elongation andΔ5-desaturation (termed the Δ6-desaturation pathway) whilst a lesscommon pathway uses a Δ9-elongation, Δ8-desaturation and Δ5-desaturation(termed the Δ9-desaturation pathway). These consecutive desaturation andelongation reactions can begin with either the ω6 fatty acid substrateLA, shown schematically as the upper left part of FIG. 1 (ω6) or the ω3substrate ALA through to EPA, shown as the lower right part of FIG. 1(ω3). If the initial Δ6-desaturation is performed on the ω6 substrateLA, the LC-PUFA product of the series of three enzymes will be the ω6fatty acid ARA. LC-PUFA synthesising organisms may convert ω6 fattyacids to ω3 fatty acids using an ω3-desaturase, shown as theΔ17-desaturase step in FIG. 1 for conversion of arachidonic acid ARA,20:4ω6) to EPA. Some members of the ω3-desaturase family can act on avariety of substrates ranging from LA to ARA. Plant ω3-desaturases oftenspecifically catalyse the Δ15-desaturation of LA to ALA, while fungaland yeast ω3-desaturases may be specific for the Δ17-desaturation of ARAto EPA (Pereira et al., 2004a; Zank et al., 2005). Some reports suggestthat non-specific ω3-desaturases may exist which can convert a widevariety of ω6 substrates to their corresponding ω3 products (Zhang etal., 2008).

The conversion of EPA to DHA in these organisms occurs by aΔ5-elongation of EPA to produce DPA, followed by a Δ4-desaturation toproduce DHA (FIG. 1). In contrast, mammals use the so-called “Sprecher”pathway which converts DPA to DHA by three separate reactions that areindependent of a Δ4-desaturase (Sprecher et al., 1995).

The front-end desaturases generally found in plants, mosses, microalgae,and lower animals such as Caenorhabditis elegans predominantly acceptfatty acid substrates esterified to the sn-2 position of aphosphatidylcholine (PC) substrate. These desaturases are thereforeknown as acyl-PC, lipid-linked, front-end desaturases (Domergue et al.,2003). In contrast, higher animal front-end desaturases generally acceptacyl-CoA substrates where the fatty acid substrate is linked to CoArather than PC (Domergue et al., 2005). Some microalgal desaturases andone plant desaturase are known to use fatty acid substrates esterifiedto CoA (Table 2).

Each PUFA elongation reaction consists of four steps catalysed by amulti-component protein complex: first, a condensation reaction resultsin the addition of a 2C unit from malonyl-CoA to the fatty acid,resulting in the formation of a β-ketoacyl intermediate. This is thenreduced by NADPH, followed by a dehydration to yield an enoylintermediate. This intermediate is finally reduced a second time toproduce the elongated fatty acid. It is generally thought that thecondensation step of these four reactions is substrate specific whilstthe other steps are not. In practice, this means that native plantelongation machinery is capable of elongating PUFA providing that thecondensation enzyme (typically called an ‘elongase’) specific to thePUFA is introduced, although the efficiency of the native plantelongation machinery in elongating the non-native PUFA substrates may below. In 2007 the identification and characterisation of the yeastelongation cycle dehydratase was published (Denic and Weissman, 2007).

PUFA desaturation in plants, mosses and microalgae naturally occurs tofatty acid substrates predominantly in the acyl-PC pool whilstelongation occurs to substrates in the acyl-CoA pool. Transfer of fattyacids from acyl-PC molecules to a CoA carrier is performed byphospholipases (PLAs) whilst the transfer of acyl-CoA fatty acids to aPC carrier is performed by lysophosphatidyl-choline acyltransferases(LPCATs) (FIG. 21) (Singh et al., 2005).

Engineered Production of LC-PUFA

Most LC-PUFA metabolic engineering has been performed using the aerobicΔ6-desaturation/elongation pathway. The biosynthesis of γ-linolenic acid(GLA, 18:3ω6) in tobacco was first reported in 1996 using aΔ6-desaturase from the cyanobacterium Synechocystis (Reddy and Thomas,1996). More recently, GLA has been produced in crop plants such assafflower (73% GLA in seedoil; Knauf et al., 2006) and soybean (28% GLA;Sato et al., 2004). The production of LC-PUFA such as EPA and DHAinvolves more complicated engineering due to the increased number ofdesaturation and elongation steps involved. EPA production in a landplant was first reported by Qi et al. (2004) who introduced genesencoding a Δ9-elongase from Isochrysis galbana, a Δ8-desaturase fromEuglena gracilis and a Δ5-desaturase from Mortierella alpina intoArabidopsis yielding up to 3% EPA. This work was followed by Abbadi etal. (2004) who reported the production of up to 0.8% EPA in flax seedusing genes encoding a Δ6-desaturase and Δ6-elongase from Physcomitrellapatens and a Δ5-desaturase from Phaeodactylum tricornutum.

The first report of DHA production, and to date the highest levels ofVLC-PUFA production reported, was in WO 04/017467 where the productionof 3% DHA in soybean embryos is described, but not seed, by introducinggenes encoding the Saprolegnia diclina Δ6-desaturase, Mortierella alpinaΔ6-desaturase, Mortierella alpina Δ5-desaturase, Saprolegnia diclinaΔ4-desaturase, Saprolegnia diclina Δ17-desaturase, Mortierella alpinaΔ6-elongase and Pavlova lutheri Δ5-elongase. The maximal EPA level inembryos also producing DHA was 19.6%, indicating that the efficiency ofconversion of EPA to DHA was poor (WO 2004/071467). This finding wassimilar to that published by Robert et al. (2005), where the flux fromEPA to DHA was low, with the production of 3% EPA and 0.5% DHA inArabidopsis using the Danio rerio Δ5/6-desaturase, the Caenorhabditiselegans Δ6-elongase, and the Pavlova salina Δ5-elongase andΔ4-desaturase. Also in 2005, Wu et al. published the production of 25%ARA, 15% EPA, and 1.5% DHA in Brassica juncea using the Pythiumirregulare Δ6-desaturase, a Thraustochytrid Δ5-desaturase, thePhyscomitrella patens Δ6-elongase, the Calendula officianalisΔ12-desaturase, a Thraustochytrid Δ5-elongase, the Phytophthorainfestans Δ17-desaturase, the Oncorhyncus mykiss LC-PUFA elongase, aThraustochytrid Δ4-desaturase and a Thraustochytrid LPCAT (Wu et al.,2005). Summaries of efforts to produce oil-seed crops which synthesizeω3 LC-PUFAs is provided in Venegas-Caleron et al. (2010) and Ruiz-Lopezet al. (2012). As indicated by Ruiz-Lopez et al. (2012), resultsobtained to date for the production of DHA in transgenic plants has beenno where near the levels seen in fish oils.

There therefore remains a need for more efficient production of LC-PUFAin recombinant cells, in particular of DHA in seeds of oilseed plants.

SUMMARY OF THE INVENTION

The present inventors have identified methods and plants for producinglipid with high levels of DHA.

In a first aspect, the present invention provides extracted plant lipid,comprising fatty acids in an esterified form, the fatty acids comprisingoleic acid, palmitic acid, ω6 fatty acids which comprise linoleic acid(LA), ω3 fatty acids which comprise α-linolenic acid (ALA), anddocosahexaenoic acid (DHA), and optionally one or more of stearidonicacid (SDA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) andeicosatetraenoic acid (ETA), wherein the level of DHA in the total fattyacid content of the extracted lipid is about 7% to 20%.

In an embodiment, the extracted lipid has one or more or all of thefollowing features

-   -   i) the level of palmitic acid in the total fatty acid content of        the extracted lipid is between about 2% and 18%, between about        2% and 16%, or between about 2% and 15%,    -   ii) the level of myristic acid (C14:0) in the total fatty acid        content of the extracted lipid is less than about 6%, less than        about 3%, less than about 2%, or less than about 1%,    -   iii) the level of oleic acid in the total fatty acid content of        the extracted lipid is between about 1% and about 30%, between        about 3% and about 30%, between about 6% and about 30%, between        1% and about 20%, between about 30% and about 60%, between about        45% to about 60%, or is about 30%,    -   iv) the level of linoleic acid (LA) in the total fatty acid        content of the extracted lipid is between about 4% and about        35%, between about 4% and about 20%, or between about 4% and        17%,    -   v) the level of α-linolenic acid (ALA) in the total fatty acid        content of the extracted lipid is between about 4% and about        40%, between about 7% and about 40%, between about 10% and about        35%, between about 20% and about 35%, between about 4% and about        16%, or between about 2% and about 16%,    -   vi) the level of γ-linolenic acid (GLA) in the total fatty acid        content of the extracted lipid is less than about 4%, less than        about 3%, less than about 2%, less than about 1%, less than        about 0.5%, between 0.05% and about 7%, between 0.05% and about        4%, between 0.05% and about 3%, or between 0.05% and about 2%,    -   vii) the level of stearidonic acid (SDA) in the total fatty acid        content of the extracted lipid is less than about 7%, less than        about 6%, less than about 4%, less than about 3%, between about        0.05% and about 7%, between about 0.05% and about 6%, between        about 0.05% and about 4%, between about 0.05% and about 3%, or        between 0.05% and about 2%,    -   viii) the level of eicosatetraenoic acid (ETA) in the total        fatty acid content of the extracted lipid is less than about 6%,        less than about 5%, less than about 4%, less than about 1%, less        than about 0.5%, between about 0.05% and about 6%, between about        0.05% and about 5%, between about 0.05% and about 4%, between        about 0.05% and about 3%, or between about 0.05% and about 2%,    -   ix) the level of eicosatrienoic acid (ETrA) in the total fatty        acid content of the extracted lipid is less than about 4%, less        than about 2%, less than about 1%, between about 0.05% and about        4%, between about 0.05% and about 3%, between about 0.05% and        about 2%, or between about 0.05% and about 1%,    -   x) the level of eicosapentaenoic acid (EPA) in the total fatty        acid content of the extracted lipid is less than about 4%, less        than about 3%, less than about 2%, between about 0.05% and about        10%, between about 0.05% and about 5%, between about 0.05% and        about 3%, or between about 0.05% and about 2%,    -   xi) the level of docosapentaenoic acid (DPA) in the total fatty        acid content of the extracted lipid is less than about 4%, less        than about 3%, less than about 2%, between about 0.05% and about        8%, between about 0.05% and about 5%, between about 0.05% and        about 3%, or between about 0.05% and about 2%,    -   xii) the level of DHA in the total fatty acid content of the        extracted lipid is about 8%, about 9%, about 10%, about 11%,        about 12%, about 13%, about 14%, about 15%, about 16%, about        17%, about 18%, between about 8% and 20%, between about 10% and        20%, between about 11% and 20%, between about 10% and about 16%,        or between about 14% and 20%,    -   xiii) the lipid comprises ω6-docosapentaenoic acid        (22:5^(Δ4, 7,10,13,16)) in its fatty acid content,    -   xiv) the lipid is essentially free of ω6-docosapentaenoic acid        (22:5^(Δ4, 7,10,13,16)) in its fatty acid content,    -   xv) the lipid is essentially free of SDA, EPA and ETA in its        fatty acid content,    -   xvi) the level of total saturated fatty acids in the total fatty        acid content of the extracted lipid is between about 4% and        about 25%, between about 4% and about 20%, between about 6% and        about 20%, between about 4% and about 60%, between about 30% and        about 60%, or between about 45% and about 60%,    -   xvii) the level of total monounsaturated fatty acids in the        total fatty acid content of the extracted lipid is between about        4% and about 35%, between about 8% and about 25%, or between 8%        and about 22%,    -   xviii) the level of total polyunsaturated fatty acids in the        total fatty acid content of the extracted lipid is between about        20% and about 75%, between about 50% and about 75%, or between        about 60% and about 75%,    -   xix) the level of total ω6 fatty acids in the total fatty acid        content of the extracted lipid is between about 35% and about        50%, between about 20% and about 35%, between about 6% and 20%,        less than about 20%, less than about 16%, less than about 10%,        between about 1% and about 16%, between about 2% and about 10%,        or between about 4% and about 10%,    -   xx) the level of new ω6 fatty acids in the total fatty acid        content of the extracted lipid is less than about 10%, less than        about 8%, less than about 6%, less than 4%, between about 1% and        about 20%, between about 1% and about 10%, between about 0.5%        and about 8%, or between about 0.5% and 4%,    -   xxi) the level of total ω3 fatty acids in the total fatty acid        content of the extracted lipid is between 36% and about 65%,        between about 40% and about 60%, between about 20% and about        35%, between about 10% and about 20%, about 25%, about 30%,        about 35% or about 40%,    -   xxii) the level of new ω3 fatty acids in the total fatty acid        content of the extracted lipid is between about 9% and about        33%, between about 10% and about 20%, between about 20% and        about 30%, between about 12% and about 25%, about 13%, about        15%, about 17% or about 20%,    -   xxiii) the ratio of total ω6 fatty acids:total ω3 fatty acids in        the fatty acid content of the extracted lipid is between about        1.0 and about 3.0, between about 0.1 and about 1, between about        0.1 and about 0.5, less than about 0.50, less than about 0.40,        less than about 0.30, less than about 0.20, less than about        0.15, about 1.0, about 0.1 or about 0.2,    -   xxiv) the ratio of new ω6 fatty acids:new ω3 fatty acids in the        fatty acid content of the extracted lipid is between about 1.0        and about 3.0, between about 0.1 and about 1, between about 0.1        and about 0.5, less than about 0.50, less than about 0.40, less        than about 0.30, less than about 0.20, less than about 0.15,        about 0.1, about 0.2 or about 1.0,    -   xxv) the fatty acid composition of the lipid is based on an        efficiency of conversion of oleic acid to LA by Δ12-desaturase        of at least about 60%, at least about 70%, at least about 80%,        between about 60% and about 98%, between about 70% and about        95%, or between about 75% and about 90%,    -   xxvi) the fatty acid composition of the lipid is based on an        efficiency of conversion of ALA to SDA by Δ6-desaturase of at        least about 30%, at least about 40%, at least about 50%, at        least about 60%, at least about 70%, between about 30% and about        70%, between about 35% and about 60%, or between about 50% and        about 70%,    -   xxvii) the fatty acid composition of the lipid is based on an        efficiency of conversion of SDA to ETA acid by Δ6-elongase of at        least about 60%, at least about 70%, at least about 75%, between        about 60% and about 95%, between about 70% and about 88%, or        between about 75% and about 85%,    -   xxviii) the fatty acid composition of the lipid is based on an        efficiency of conversion of ETA to EPA by Δ5-desaturase of at        least about 60%, at least about 70%, at least about 75%, between        about 60% and about 99%, between about 70% and about 99%, or        between about 75% and about 98%,    -   xxix) the fatty acid composition of the lipid is based on an        efficiency of conversion of EPA to DPA by Δ5-elongase of at        least about 80%, at least about 85%, at least about 90%, between        about 50% and about 95%, or between about 85% and about 95%,    -   xxx) the fatty acid composition of the lipid is based on an        efficiency of conversion of DPA to DHA by Δ4-desaturase of at        least about 80%, at least about 90%, at least about 93%, between        about 50% and about 95%, between about 80% and about 95%, or        between about 85% and about 95%,    -   xxxi) the fatty acid composition of the lipid is based on an        efficiency of conversion of oleic acid to DHA of at least about        10%, at least about 15%, at least about 20%, between about 10%        and about 50%, between about 10% and about 30%, or between about        10% and about 25%,    -   xxxii) the fatty acid composition of the lipid is based on an        efficiency of conversion of LA to DHA of at least about 15%, at        least about 20%, at least about 22%, at least about 25%, between        about 15% and about 50%, between about 20% and about 40%, or        between about 20% and about 30%,    -   xxxiii) the fatty acid composition of the lipid is based on an        efficiency of conversion of ALA to DHA of at least about 17%, at        least about 22%, at least about 24%, between about 17% and about        55%, between about 22% and about 35%, or between about 24% and        about 35%,    -   xxxiv) the total fatty acid in the extracted lipid has less than        1% C20:1,    -   xxxv) the triacylglycerol (TAG) content of the lipid is at least        about 70%, at least about 80%, at least about 90%, at least 95%,        between about 70% and about 99%, or between about 90% and about        99%,    -   xxxvi) the lipid comprises diacylglycerol (DAG),    -   xxxvii) the lipid comprises less than about 10%, less than about        5%, less than about 1%, or between about 0.001% and about 5%,        free (non-esterified) fatty acids and/or phospholipid, or is        essentially free thereof,    -   xxxviii) at least 70%, or at least 80%, of the DHA esterified in        the form of TAG is in the sn-1 or sn-3 position of the TAG,    -   xxxix) the most abundant DHA-containing TAG species in the lipid        is DHA/18:3/18:3 (TAG 58:12), and    -   xl) the lipid comprises tri-DHA TAG (TAG 66:18).

In another embodiment, the extracted lipid is in the form of an oil,wherein at least about 90%, or least about 95%, at least about 98%, orbetween about 95% and about 98%, by weight of the oil is the lipid.

In a preferred embodiment, the lipid or oil, preferably a seedoil, hasthe following features: in the total fatty acid content of the lipid oroil, the level of DHA is between about 7% and 20%, the level of palmiticacid is between about 2% and about 16%, the level of myristic acid isless than about 6%, the level of oleic acid is between about 1% andabout 30%, the level of LA is between about 4% and about 35%, ALA ispresent, GLA is present, the level of SDA is between about 0.05% andabout 7%, the level of ETA is less than about 4%, the level of EPA isbetween about 0.05% and about 10%, the level of DPA is between about0.05% and about 8%, the level of total saturated fatty acids in thetotal fatty acid content of the extracted lipid is between about 4% andabout 25%, the level of total monounsaturated fatty acids in the totalfatty acid content of the extracted lipid is between about 4% and about35%, the level of total polyunsaturated fatty acids in the total fattyacid content of the extracted lipid is between about 20% and about 75%,the ratio of total ω6 fatty acids:total ω3 fatty acids in the fatty acidcontent of the extracted lipid is between about 0.05 and about 3.0, theratio of new ω6 fatty acids:new ω3 fatty acids in the fatty acid contentof the extracted lipid is between about 0.03 and about 3.0, preferablyless than about 0.50, the fatty acid composition of the lipid is basedon: an efficiency of conversion of oleic acid to LA by Δ12-desaturase ofat least about 60%, an efficiency of conversion of SDA to ETA acid byΔ6-elongase of at least about 60%, an efficiency of conversion of EPA toDPA by Δ5-elongase of between about 50% and about 95%, an efficiency ofconversion of DPA to DHA by Δ4-desaturase of between about 50% and about95%, an efficiency of conversion of oleic acid to DHA of at least about10%, and the triacylglycerol (TAG) content of the lipid is at leastabout 70%, and optionally the lipid is essentially free of cholesteroland/or the lipid comprises tri-DHA TAG (TAG 66:18).

In a more preferred embodiment, the lipid or oil, preferably a seedoil,has the following features: in the total fatty acid content of thelipid, the level of DHA is between about 7% and 20%, the level ofpalmitic acid is between about 2% and about 16%, the level of myristicacid is less than about 2%, the level of oleic acid is between about 1%and about 30%, the level of LA is between about 4% and about 35%, thelevel of ALA is between about 7% and about 40%, the level of GLA is lessthan about 4%, the level of SDA is between about 0.05% and about 7%, thelevel of ETA is less than about 4%, the level of ETrA is between about0.05% and about 4%, the level of EPA is between about 0.05% and about10%, the level of DPA is between about 0.05% and about 8%, the level oftotal saturated fatty acids in the total fatty acid content of theextracted lipid is between about 4% and about 25%, the level of totalmonounsaturated fatty acids in the total fatty acid content of theextracted lipid is between about 4% and about 35%, the level of totalpolyunsaturated fatty acids in the total fatty acid content of theextracted lipid is between about 20% and about 75%, the level of new ω6fatty acids in the total fatty acid content of the extracted lipid isbetween about 0.5% and about 10%, the level of total ω3 fatty acids inthe total fatty acid content of the extracted lipid is between 36% andabout 75%, the level of new ω3 fatty acids in the total fatty acidcontent of the extracted lipid is between about 9% and about 33%, theratio of total ω6 fatty acids:total ω3 fatty acids in the fatty acidcontent of the extracted lipid is between about 0.05 and about 3.0, theratio of new ω6 fatty acids:new ω3 fatty acids in the fatty acid contentof the extracted lipid is between about 0.03 and about 3.0, the fattyacid composition of the lipid is based on: an efficiency of conversionof oleic acid to LA by Δ12-desaturase of at least about 60%, anefficiency of conversion of SDA to ETA acid by Δ6-elongase of at leastabout 60%, an efficiency of conversion of ETA to EPA by Δ5-desaturase ofat least about 60%, an efficiency of conversion of EPA to DPA byΔ5-elongase of between about 50% and about 95%, an efficiency ofconversion of DPA to DHA by Δ4-desaturase of between about 50% and about95%, an efficiency of conversion of oleic acid to DHA of at least about10%, an efficiency of conversion of LA to DHA of at least about 15%, anefficiency of conversion of ALA to DHA of at least about 17%, and thetotal fatty acid content in the extracted lipid has less than 1% C20:1,the triacylglycerol (TAG) content of the lipid is at least about 70%,the lipid is essentially free of cholesterol, and the lipid comprisestri-DHA TAG (TAG 66:18). Preferably, the lipid or oil is canola oiland/or has not been treated with a transesterification process after itwas extracted from the plant or plant part. In a particular embodiment,the lipid or canola oil may subsequently be treated to convert the fattyacids in the oil to alkyl esters such as methyl or ethyl esters. Furthertreatment may be applied to enrich the lipid or oil for the DHA.

In an embodiment, the lipid or oil, preferably a seedoil, has thefollowing features: in the total fatty acid content of the lipid, thelevel of DHA is between about 7% and 20%, the level of palmitic acid isbetween about 2% and about 16%, the level of myristic acid is less thanabout 2%, the level of oleic acid is between about 30% and about 60%,preferably between about 45% and about 60%, the level of LA is betweenabout 4% and about 20%, the level of ALA is between about 2% and about16%, the level of GLA is less than about 3%, the level of SDA is lessthan about 3%, the level of ETA is less than about 4%, the level of ETrAless than about 2%, the level of EPA is less than about 4%, the level ofDPA is less than about 4%, the level of total saturated fatty acids inthe total fatty acid content of the extracted lipid is between about 4%and about 25%, the level of total monounsaturated fatty acids in thetotal fatty acid content of the extracted lipid is between about 30% andabout 60%, or between about 40% and about 60%, the level of totalpolyunsaturated fatty acids in the total fatty acid content of theextracted lipid is between about 20% and about 75%, the level of new ω6fatty acids in the total fatty acid content of the extracted lipid isbetween about 0.5% and about 10%, the level of total ω3 fatty acids inthe total fatty acid content of the extracted lipid is between about 10%and about 20%, the level of new ω3 fatty acids in the total fatty acidcontent of the extracted lipid is between about 9% and about 20%, theratio of total ω6 fatty acids:total ω3 fatty acids in the fatty acidcontent of the extracted lipid is between about 0.05 and about 3.0,preferably less than about 0.50, the ratio of new ω6 fatty acids:new ω3fatty acids in the fatty acid content of the extracted lipid is betweenabout 0.03 and about 3.0, the triacylglycerol (TAG) content of the lipidis at least about 70%, the lipid is essentially free of cholesterol, andthe lipid comprises tri-DHA TAG (TAG 66:18). Preferably, the lipid oroil is essentially free of SDA, EPA and ETA and/or is canola oil and/orhas not been treated with a transesterification process after it wasextracted from the plant or plant part. In a particular embodiment, thelipid or canola oil may subsequently be treated to convert the fattyacids in the oil to alkyl esters such as methyl or ethyl esters. Furthertreatment may be applied to enrich the lipid or oil for the DHA.

In a further preferred embodiment, the lipid or oil, preferably aseedoil, has the following features: in the total fatty acid content ofthe lipid or oil, the level of DHA is between about 7% and 20%, thelevel of palmitic acid is between about 2% and about 16%, the level ofmyristic acid is less than about 6%, the level of oleic acid is betweenabout 1% and about 30%, the level of LA is between about 4% and about35%, ALA is present, GLA is present, the level of SDA is between about0.05% and about 7%, the level of ETA is less than about 6%, the level ofEPA is between about 0.05% and about 10%, the level of DPA is betweenabout 0.05% and about 8%.

In a further embodiment, the extracted lipid further comprises one ormore sterols, preferably plant sterols.

In another embodiment, the extracted lipid is in the form of an oil, andcomprises less than about 10 mg of sterols/g of oil, less than about 7mg of sterols/g of oil, between about 1.5 mg and about 10 mg ofsterols/g of oil, or between about 1.5 mg and about 7 mg of sterols/g ofoil.

Examples of sterols which can be in the extracted lipid include, but arenot necessarily limited to, one or more or all ofcampesterol/24-methylcholesterol, Δ5-stigmasterol, eburicol,β-sitosterol/24-ethylcholesterol, Δ5-avenasterol/isofucosterol,Δ7-stigmasterol/stigmast-7-en-3β-ol, and Δ7-avenasterol.

In an embodiment, the plant species is one listed in Table 26, such ascanola, and the level of sterols are about the same as that listed inTable 26 for that particular plant species.

In an embodiment, the extracted lipid comprises less than about 0.5 mgof cholesterol/g of oil, less than about 0.25 mg of cholesterol/g ofoil, between about 0 mg and about 0.5 mg of cholesterol/g of oil, orbetween about 0 mg and about 0.25 mg of cholesterol/g of oil, or whichis essentially free of cholesterol.

In a further embodiment, the lipid is an oil, preferably oil from anoilseed. Examples of such oils include, but are not limited to, Brassicasp. oil such as canola oil, Gossypium hirsutum oil, Linum usitatissimumoil, Helianthus sp. oil, Carthamus tinctorius oil, Glycine max oil, Zeamays oil, Arabidopsis thaliana oil, Sorghum bicolor oil, Sorghum vulgareoil, Avena sativa oil, Trifolium sp. oil, Elaesis guineenis oil,Nicotiana benthamiana oil, Hordeum vulgare oil, Lupinus angustifoliusoil, Oryza sativa oil, Oryza glaberrima oil, Camelina sativa oil, Crambeabyssinica oil, Miscanthus×giganteus oil, or Miscanthus sinensis oil.

Also provided is extracted plant lipid, preferably extracted canolaseedoil, comprising fatty acids in an esterified form, the fatty acidscomprising oleic acid, palmitic acid, ω6 fatty acids which compriselinoleic acid (LA), ω3 fatty acids which comprise α-linolenic acid(ALA), and docosahexaenoic acid (DHA), and optionally one or more ofstearidonic acid (SDA), eicosapentaenoic acid (EPA), docosapentaenoicacid (DPA) and eicosatetraenoic acid (ETA), wherein lipid has thefollowing features in the total fatty acid content of the lipid;

i) the level of DHA is about 3%, about 4%, about 5%, about 6% or about7%,

ii) the level of palmitic acid is between about 2% and about 16%,

iii) the level of myristic acid is less than about 2%,

iv) the level of oleic acid is between about 30% and about 60%,preferably between about 45% and about 60%,

v) the level of LA is between about 4% and about 20%,

vi) the level of ALA is between about 2% and about 16%,

vii) the level of GLA is less than about 4%,

viii) the level of SDA is less than about 6%, or less than about 4%,

ix) the level of ETA is less than about 6%, or less than about 4%,

x) the level of ETrA less than about 1%,

xi) the level of EPA is less than about 10% and/or the level of EPA is0.5-2.0 fold the level of DHA,

xii) the level of DPA is less than about 4%,

xiii) the level of total saturated fatty acids in the total fatty acidcontent of the extracted lipid is between about 4% and about 25%,

xiv) the level of total monounsaturated fatty acids in the total fattyacid content of the extracted lipid is between about 30% and about 70%,

xv) the level of total polyunsaturated fatty acids in the total fattyacid content of the extracted lipid is between about 15% and about 75%,preferably between about 15% and about 30%,

xvi) the level of new ω6 fatty acids in the total fatty acid content ofthe extracted lipid is between about 0.5% and about 10%,

xvii) the level of total ω3 fatty acids in the total fatty acid contentof the extracted lipid is between about 10% and about 20%,

xviii) the level of new ω3 fatty acids in the total fatty acid contentof the extracted lipid is between about 3% and about 20%,

xix) the ratio of total ω6 fatty acids:total ω3 fatty acids in the fattyacid content of the extracted lipid is between about 0.05 and about 3.0,preferably less than about 0.50,

xx) the ratio of new ω6 fatty acids:new ω3 fatty acids in the fatty acidcontent of the extracted lipid is between about 0.03 and about 3.0,

xxi) the triacylglycerol (TAG) content of the lipid is at least about70%, and

xxii) the lipid is essentially free of cholesterol. In an embodiment,the lipid comprises tri-DHA TAG (TAG 66:18). More preferably, the lipidis essentially free of SDA and ETA, and/or has not been treated with atransesterification process after it was extracted from the plant orplant part.

In another aspect, provided is extracted plant lipid, comprising fattyacids in an esterified form, the fatty acids comprising oleic acid,palmitic acid, ω6 fatty acids which comprise linoleic acid (LA), ω3fatty acids which comprise α-linolenic acid (ALA) and docosahexaenoicacid (DHA), and one or more of stearidonic acid (SDA), eicosapentaenoicacid (EPA), docosapentaenoic acid (DPA) and eicosatetraenoic acid (ETA),wherein (i) the level of DHA in the total fatty acid content of theextracted lipid is between 7% and 20%, (ii) the level of palmitic acidin the total fatty acid content of the extracted lipid is between 2% and16%, (iii) the level of myristic acid (C14:0) in the total fatty acidcontent of the extracted lipid is less than 6%, (iv) the level of oleicacid in the total fatty acid content of the extracted lipid is between1% and 30% or between 30% and 60%, (v) the level of linoleic acid (LA)in the total fatty acid content of the extracted lipid is between 4% and35%, (vi) the level of α-linolenic acid (ALA) in the total fatty acidcontent of the extracted lipid is between 4% and 40%, (vii) the level ofeicosatrienoic acid (ETrA) in the total fatty acid content of theextracted lipid is less than 4%, (viii) the level of total saturatedfatty acids in the total fatty acid content of the extracted lipid isbetween 4% and 25%, (ix) the ratio of total ω6 fatty acids:total ω3fatty acids in the fatty acid content of the extracted lipid is between1.0 and 3.0 or between 0.1 and 1, (x) the triacylglycerol (TAG) contentof the lipid is at least 70%, and (xi) at least 70% of the DHAesterified in the form of TAG is in the sn-1 or sn-3 position of theTAG. In an embodiment, one or more or all of the following features

-   -   i) the level of palmitic acid in the total fatty acid content of        the extracted lipid is between 2% and 15%,    -   ii) the level of myristic acid (C14:0) in the total fatty acid        content of the extracted lipid is less than 1%,    -   iii) the level of oleic acid in the total fatty acid content of        the extracted lipid is between about 3% and about 30%, between        about 6% and about 30%, between 1% and about 20%, between about        45% and about 60%, or is about 30%,    -   iv) the level of linoleic acid (LA) in the total fatty acid        content of the extracted lipid is between about 4% and about        20%, or between about 4% and 17%,    -   v) the level of t-linolenic acid (ALA) in the total fatty acid        content of the extracted lipid is between about 7% and about        40%, between about 10% and about 35%, between about 20% and        about 35%, or between about 4% and 16%,    -   vi) the level of γ-linolenic acid (GLA) in the total fatty acid        content of the extracted lipid is less than 4%, less than about        3%, less than about 2%, less than about 1%, less than about        0.5%, between 0.05% and 7%, between 0.05% and 4%, or between        0.05% and about 3%, or between 0.05% and about 2%,    -   vii) the level of stearidonic acid (SDA) in the total fatty acid        content of the extracted lipid is less than about 4%, less than        about 3%, between about 0.05% and about 7%, between about 0.05%        and about 4%, between about 0.05% and about 3%, or between 0.05%        and about 2%,    -   viii) the level of eicosatetraenoic acid (ETA) in the total        fatty acid content of the extracted lipid is less than about 4%,        less than about 1%, less than about 0.5%, between about 0.05%        and about 5%, between about 0.05% and about 4%, between about        0.05% and about 3%, or between about 0.05% and about 2%,    -   ix) the level of eicosatrienoic acid (ETrA) in the total fatty        acid content of the extracted lipid is less than about 2%, less        than about 1%, between 0.05% and 4%, between 0.05% and 3%, or        between 0.05% and about 2%, or between 0.05% and about 1%,    -   x) the level of eicosapentaenoic acid (EPA) in the total fatty        acid content of the extracted lipid is less than 4%, less than        about 3%, less than about 2%, between 0.05% and 10%, between        0.05% and 5%, or between 0.05% and about 3%, or between 0.05%        and about 2%,    -   xi) the level of docosapentaenoic acid (DPA) in the total fatty        acid content of the extracted lipid is less than 4%, less than        about 3%, less than about 2%, between 0.05% and 8%, between        0.05% and 5%, or between 0.05% and about 3%, or between 0.05%        and about 2%,    -   xii) the level of DHA in the total fatty acid content of the        extracted lipid is about 8%, about 9%, about 10%, about 11%,        about 12%, about 13%, about 14%, about 15%, about 16%, about        17%, about 18%, between about 8% and 20%, between about 10% and        20%, between about 11% and 20%, between about 10% and about 16%,        or between about 14% and 20%,    -   xiii) the lipid comprises ω6-docosapentaenoic acid        (22:5^(Δ4,7,10,13,16)) in its fatty acid content,    -   xiv) the lipid is essentially free of ω6-docosapentaenoic acid        (22:5^(Δ4,7,10,13,16)) in its fatty acid content,    -   xv) the lipid is essentially free of SDA, EPA and ETA in its        fatty acid content,    -   xvi) the level of total saturated fatty acids in the total fatty        acid content of the extracted lipid is between about 4% and        about 20%, or between about 6% and about 20%,    -   xvii) the level of total monounsaturated fatty acids in the        total fatty acid content of the extracted lipid is between about        4% and about 35%, between about 8% and about 25%, or between 8%        and about 22%,    -   xviii) the level of total polyunsaturated fatty acids in the        total fatty acid content of the extracted lipid is between about        20% and about 75%, between about 50% and about 75%, or between        about 60% and about 75%,    -   xix) the level of total ω6 fatty acids in the total fatty acid        content of the extracted lipid is between about 35% and about        50%, between about 20% and about 35%, between about 6% and 20%,        less than 20%, less than about 16%, less than about 10%, between        about 1% and about 16%, between about 2% and about 10%, or        between about 4% and about 10%,    -   xx) the level of new ω6 fatty acids in the total fatty acid        content of the extracted lipid is less than about 10%, less than        about 8%, less than about 6%, less than 4%, between about 1% and        about 20%, between about 1% and about 10%, between about 0.5%        and about 8%, or between about 0.5% and 4%,    -   xxi) the level of total ω3 fatty acids in the total fatty acid        content of the extracted lipid is between 36% and about 65%,        between 40% and about 60%, between about 20% and about 35%,        between about 10% and about 20%, about 25%, about 30%, about 35%        or about 40%,    -   xxii) the level of new ω3 fatty acids in the total fatty acid        content of the extracted lipid is between 9% and about 33%,        between about 10% and about 20%, between about 20% and about        30%, between about 12% and about 25%, about 13%, about 15%,        about 17% or about 20%,    -   xxiii) the ratio of total ω6 fatty acids:total ω3 fatty acids in        the fatty acid content of the extracted lipid is between about        0.1 and about 0.5, less than about 0.50, less than about 0.40,        less than about 0.30, less than about 0.20, less than about        0.15, about 1.0, about 0.1 or about 0.2,    -   xxiv) the ratio of new ω6 fatty acids:new ω3 fatty acids in the        fatty acid content of the extracted lipid is between about 1.0        and about 3.0, between about 0.1 and about 1, between about 0.1        and about 0.5, less than about 0.50, less than about 0.40, less        than about 0.30, less than about 0.20, less than about 0.15,        about 0.1, about 0.2 or about 1.0,    -   xxv) the fatty acid composition of the lipid is based on an        efficiency of conversion of oleic acid to DHA of at least about        10%, at least about 15%, at least about 20%, between about 10%        and about 50%, between about 10% and about 30%, or between about        10% and about 25%,    -   xxvi) the fatty acid composition of the lipid is based on an        efficiency of conversion of LA to DHA of at least about 15%, at        least about 20%, at least about 22%, at least about 25%, between        about 15% and about 50%, between about 20% and about 40%, or        between about 20% and about 30%,    -   xxvii) the fatty acid composition of the lipid is based on an        efficiency of conversion of ALA to DHA of at least about 17%, at        least about 22%, at least about 24%, between about 17% and about        55%, between about 22% and about 35%, or between about 24% and        about 35%,    -   xxviii) the total fatty acid in the extracted lipid has less        than 1% C20:1,    -   xxix) the triacylglycerol (TAG) content of the lipid is at least        about 80%, at least about 90%, at least 95%, between about 70%        and about 99%, or between about 90% and about 99%,    -   xxx) the lipid comprises diacylglycerol (DAG),    -   xxxi) the lipid comprises less than about 10%, less than about        5%, less than about 1%, or between about 0.001% and about 5%,        free (non-esterified) fatty acids and/or phospholipid, or is        essentially free thereof,    -   xxxii) at least 80%, of the DHA esterified in the form of TAG is        in the sn-1 or sn-3 position of the TAG,    -   xxxiii) the most abundant DHA-containing TAG species in the        lipid is DHA/18:3/18:3 (TAG 58:12), and    -   xxxiv) the lipid comprises tri-DHA TAG (TAG 66:18).

With specific regard to the above aspect, in an embodiment

i) the lipid is in the form of an oil, wherein the oil comprises one ormore sterols such as one or more or all of campesterol, Δ5-stigmasterol,eburicol, β-sitosterol, Δ5-avenasterol, Δ7-stigmasterol andΔ7-avenasterol, and optionally the oil comprises less than 10 mg ofsterols/g of oil and/or the oil is essentially free of cholesterol,and/or

ii) the lipid is in the form of an oil from an oilseed such as oilseedis a Brassica sp oilseed or canola seed.

In another aspect, the present invention provides a process forproducing extracted plant lipid, comprising the steps of

i) obtaining a plant part comprising lipid, the lipid comprising fattyacids in an esterified form, the fatty acids comprising oleic acid,palmitic acid, ω6 fatty acids which comprise linoleic acid (LA), ω3fatty acids which comprise t-linolenic acid (ALA), and docosahexaenoicacid (DHA), and optionally one or more of eicosapentaenoic acid (EPA),stearidonic acid (SDA), docosapentaenoic acid (DPA) and eicosatetraenoicacid (ETA), wherein the level of DHA in the total fatty acid content ofextractable lipid in the plant part is about 7% to 20%, and

ii) extracting lipid from the plant part, wherein the level of DHA inthe total fatty acid content of the extracted lipid is about 7% to 20%.

In a preferred embodiment, the extracted lipid has one or more of thefeatures defined above.

In an embodiment, wherein the plant part is a seed, preferably anoilseed. Examples of such seeds include, but are not limited to,Brassica sp., Gossypium hirsutum, Linum usitatissimum, Helianthus sp.,Carthamus tinctorius, Glycine max, Zea mays, Arabidopsis thaliana,Sorghum bicolor, Sorghum vulgare, Avena sativa, Trifolium sp., Elaesisguineenis, Nicotiana benthamiana, Hordeum vulgare, Lupinusangustifolius, Oryza sativa, Oryza glaberrima, Camelina sativa, orCrambe abyssinica, preferably a Brassica napus, B. juncea or C. sativaseed.

In another embodiment, the seed comprises at least about 18 mg, at leastabout 22 mg, at least about 26 mg, between about 18 mg and about 100 mg,between about 22 mg and about 70 mg, or between about 24 mg and about 50mg, of DHA per gram of seed.

In a further embodiment, the plant part comprises exogenouspolynucleotides encoding one of the following sets of enzymes;

i) an ω3-desaturase, a Δ6-desaturase, a Δ5-desaturase, a Δ4-desaturase,a Δ6-elongase and a Δ5-elongase,

ii) a Δ15-desaturase, a Δ6-desaturase, a Δ5-desaturase, a Δ4-desaturase,a Δ6-elongase and a Δ5-elongase,

iii) a Δ12-desaturase, a Δ6-desaturase, a Δ5-desaturase, aΔ4-desaturase, a Δ6-elongase and an Δ5-elongase,

iv) a Δ12-desaturase, a ω3-desaturase or a Δ15-desaturase, aΔ6-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ6-elongase and anΔ5-elongase,

v) an ω3-desaturase, a Δ8-desaturase, a Δ5-desaturase, a Δ4-desaturase,a Δ9-elongase and an Δ5-elongase,

vi) a Δ15-desaturase, a Δ8-desaturase, a Δ5-desaturase, a Δ4-desaturase,a Δ9-elongase and a Δ5-elongase,

vii) a Δ12-desaturase, a Δ8-desaturase, a Δ5-desaturase, aΔ4-desaturase, a Δ9-elongase and an Δ5-elongase, or

viii) a Δ12-desaturase, a ω3-desaturase or a Δ15-desaturase, aΔ8-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ9-elongase and anΔ5-elongase, and wherein each polynucleotide is operably linked to oneor more promoters that are capable of directing expression of saidpolynucleotides in a cell of the plant part.

In yet a further embodiment, the plant part has one or more or all ofthe following features

i) the Δ12-desaturase converts oleic acid to linoleic acid in one ormore cells of the plant with an efficiency of at least about 60%, atleast about 70%, at least about 80%, between about 60% and about 98%,between about 70% and about 95%, or between about 75% and about 90%,

ii) the ω3-desaturase converts ω6 fatty acids to ω3 fatty acids in oneor more cells of the plant with an efficiency of at least about 65%, atleast about 75%, at least about 85%, between about 65% and about 95%,between about 75% and about 95%, or between about 80% and about 95%,

iii) the Δ6-desaturase converts ALA to SDA in one or more cells of theplant with an efficiency of at least about 30%, at least about 40%, atleast about 50%, at least about 60%, at least about 70%, between about30% and about 70%, between about 35% and about 60%, or between about 50%and about 70%,

iv) the Δ6-desaturase converts linoleic acid to γ-linolenic acid in oneor more cells of the plant with an efficiency of less than about 5%,less than about 2.5%, less than about 1%, between about 0.1% and about5%, between about 0.5% and about 2.5%, or between about 0.5% and about1%,

v) the Δ6-elongase converts SDA to ETA in one or more cells of the plantwith an efficiency of at least about 60%, at least about 70%, at leastabout 75%, between about 60% and about 95%, between about 70% and about88%, or between about 75% and about 85%,

vi) the Δ5-desaturase converts ETA to EPA in one or more cells of theplant with an efficiency of at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 90%, between about60% and about 99%, between about 70% and about 99%, or between about 75%and about 98%,

vii) the Δ5-elongase converts EPA to DPA in one or more cells of theplant with an efficiency of at least about 80%, at least about 85%, atleast about 90%, between about 50% and about 95%, or between about 85%and about 95%,

viii) the Δ4-desaturase converts DPA to DHA in one or more cells of theplant with an efficiency of at least about 80%, at least about 90%, atleast about 93%, between about 50% and about 95%, between about 80% andabout 95%, or between about 85% and about 95%,

ix) the efficiency of conversion of oleic acid to DHA in one or morecells of the plant part is at least about 10%, at least about 15%, atleast about 20%, between about 10% and about 50%, between about 10% andabout 30%, or between about 10% and about 25%,

x) the efficiency of conversion of LA to DHA in one or more cells of theplant part is at least about 15%, at least about 20%, at least about22%, at least about 25%, between about 15% and about 50%, between about20% and about 40%, or between about 20% and about 30%,

xi) the efficiency of conversion of ALA to DHA in one or more cells ofthe plant part is at least about 17%, at least about 22%, at least about24%, between about 17% and about 55%, between about 22% and about 35%,or between about 24% and about 35%,

xii) one or more cells of the plant part comprise at least about 15%, atleast about 20%, between about 15% and about 30%, or between about 22.5%and about 27.5%, more ω3 fatty acids than corresponding cells lackingthe exogenous polynucleotides,

xiii) the Δ6-desaturase preferentially desaturates t-linolenic acid(ALA) relative to linoleic acid (LA),

xiv) the Δ6-elongase also has Δ9-elongase activity,

xv) the Δ12-desaturase also has Δ15-desaturase activity,

xvi) the Δ6-desaturase also has Δ8-desaturase activity,

xvii) the Δ8-desaturase also has Δ6-desaturase activity or does not haveΔ6-desaturase activity,

xviii) the Δ15-desaturase also has ω3-desaturase activity on GLA,

xix) the ω3-desaturase also has Δ15-desaturase activity on LA,

xx) the ω3-desaturase desaturates both LA and/or GLA,

xxi) the ω3-desaturase preferentially desaturates GLA relative to LA,

xxii) the level of DHA in the plant part is based on an efficiency ofconversion of oleic acid to DHA in the plant part of at least about 10%,at least about 15%, at least about 20%, between about 10% and about 50%,between about 15% and about 30%, or between about 20% and about 25%,

xxiii) the level of DHA in the plant part is based on an efficiency ofconversion of LA to DHA in the plant part of at least about 15%, atleast about 20%, at least about 22%, between about 15% and about 60%,between about 20% and about 40%, or between about 22% and about 30%,

xxiv) the level of DHA in the plant part is based on an efficiency ofconversion of ALA to DHA in the plant part of at least about 17%, atleast about 22%, at least about 24%, between about 17% and about 65%,between about 22% and about 35%, or between about 24% and about 35%

xxx) one or more or all of the desaturases have greater activity on anacyl-CoA substrate than a corresponding acyl-PC substrate,

xxxi) the Δ6-desaturase has greater Δ6-desaturase activity on ALA thanLA as fatty acid substrate,

xxxii) the Δ6-desaturase has greater Δ6-desaturase activity on ALA-CoAas fatty acid substrate than on ALA joined to the sn-2 position of PC asfatty acid substrate,

xxxiii) the Δ6-desaturase has at least about a 2-fold greaterΔ6-desaturase activity, at least 3-fold greater activity, at least4-fold greater activity, or at least 5-fold greater activity, on ALA asa substrate compared to LA,

xxxiv) the Δ6-desaturase has greater activity on ALA-CoA as fatty acidsubstrate than on ALA joined to the sn-2 position of PC as fatty acidsubstrate,

xxxv) the Δ6-desaturase has at least about a 5-fold greaterΔ6-desaturase activity or at least 10-fold greater activity, on ALA-CoAas fatty acid substrate than on ALA joined to the sn-2 position of PC asfatty acid substrate,

xxxvi) the desaturase is a front-end desaturase,

xxxvii) the Δ6-desaturase has no detectable Δ5-desaturase activity onETA.

In yet a further embodiment, the plant part has one or more or all ofthe following features

i) the Δ12-desaturase comprises amino acids having a sequence asprovided in SEQ ID NO:10, a biologically active fragment thereof, or anamino acid sequence which is at least 50% identical to SEQ ID NO:10,

ii) the ω3-desaturase comprises amino acids having a sequence asprovided in SEQ ID NO:12, a biologically active fragment thereof, or anamino acid sequence which is at least 50% identical to SEQ ID NO:12,

iii) the Δ6-desaturase comprises amino acids having a sequence asprovided in SEQ ID NO:16, a biologically active fragment thereof, or anamino acid sequence which is at least 50% identical to SEQ ID NO:16,

iv) the Δ6-elongase comprises amino acids having a sequence as providedin SEQ ID NO:25, a biologically active fragment thereof such as SEQ IDNO:26, or an amino acid sequence which is at least 50% identical to SEQID NO:25 and/or SEQ ID NO:26,

v) the Δ5-desaturase comprises amino acids having a sequence as providedin SEQ ID NO:30, a biologically active fragment thereof, or an aminoacid sequence which is at least 50% identical to SEQ ID NO:30,

vi) the Δ5-elongase comprises amino acids having a sequence as providedin SEQ ID NO:37, a biologically active fragment thereof, or an aminoacid sequence which is at least 50% identical to SEQ ID NO:37,

vii) the Δ4-desaturase comprises amino acids having a sequence asprovided in SEQ ID NO:41, a biologically active fragment thereof, or anamino acid sequence which is at least 50% identical to SEQ ID NO:41.

In an embodiment, the plant part further comprises an exogenouspolynucleotide encoding a diacylglycerol acyltransferase (DGAT),monoacylglycerol acyltransferase (MGAT), glycerol-3-phosphateacyltransferase (GPAT), 1-acyl-glycerol-3-phosphate acyltransferase(LPAAT) preferably an LPAAT which can use a C22 polyunsaturated fattyacyl-CoA substrate, acyl-CoA:lysophosphatidylcholine acyltransferase(LPCAT), phospholipase A₂ (PLA₂), phospholipase C (PLC), phospholipase D(PLD), CDP-choline diacylglycerol choline phosphotransferase (CPT),phoshatidylcholine diacylglycerol acyltransferase (PDAT),phosphatidylcholine:diacylglycerol choline phosphotransferase (PDCT),acyl-CoA synthase (ACS), or a combination of two or more thereof.

In another embodiment, the plant part further comprises an introducedmutation or an exogenous polynucleotide which down regulates theproduction and/or activity of an endogenous enzyme in a cell of theplant part selected from FAE1, DGAT, MGAT, GPAT, LPAAT, LPCAT, PLA₂,PLC, PLD, CPT, PDAT, a thioesterase such as FATB, or a Δ12-desaturase,or a combination of two or more thereof.

In a further embodiment, at least one, or all, of the promoters are seedspecific promoters. In an embodiment, at least one, or all, of thepromoters have been obtained from oil biosynthesis or accumulation genessuch as oleosin, or from seed storage protein genes such as conlinin.

In another embodiment, the promoter(s) directing expression of theexogenous polynucleotides encoding the Δ4-desaturase and the Δ5-elongaseinitiate expression of the polynucleotides in developing seed of theplant part before, or reach peak expression before, the promoter(s)directing expression of the exogenous polynucleotides encoding theΔ12-desaturase and the ω3-desaturase.

In a further embodiment, the exogenous polynucleotides are covalentlylinked in a DNA molecule, preferably a T-DNA molecule, integrated intothe genome of cells of the plant part and preferably where the number ofsuch DNA molecules integrated into the genome of the cells of the plantpart is not more than one, two or three, or is two or three.

In yet another embodiment, the plant comprises at least two different,exogenous polynucleotides each encoding a Δ6-desaturase which have thesame or different amino acid sequences.

In a further embodiment, the total oil content of the plant partcomprising the exogenous polynucleotides is at least about 40%, or atleast about 50%, or at least about 60%, or at least about 70%, orbetween about 50% and about 80% of the total oil content of acorresponding plant part lacking the exogenous polynucleotides. In theseembodiments, the maximum oil content may be about 100% of the oilcontent of a corresponding wild-type plant part.

In another embodiment, the lipid is in the form of an oil, preferably aseedoil from an oilseed, and wherein at least about 90%, or about least95%, at least about 98%, or between about 95% and about 98%, by weightof the lipid is triacylglycerols.

In a further embodiment, the process further comprises treating thelipid to increase the level of DHA as a percentage of the total fattyacid content. For example, the treatment is transesterification. Forexample, the lipid such as canola oil may be treated to convert thefatty acids in the oil to alkyl esters such as methyl or ethyl esters,which may then be fractionated to enrich the lipid or oil for the DHA.

Further, provided is a process for producing extracted plant lipid,comprising the steps of

i) obtaining a plant part, preferably canola seed, comprising lipid, thelipid comprising fatty acids in an esterified form, the fatty acidscomprising oleic acid, palmitic acid, ω6 fatty acids which compriselinoleic acid (LA), ω3 fatty acids which comprise α-linolenic acid(ALA), and docosahexaenoic acid (DHA), and optionally one or more ofeicosapentaenoic acid (EPA), stearidonic acid (SDA), docosapentaenoicacid (DPA) and eicosatetraenoic acid (ETA), wherein the level of DHA inthe total fatty acid content of extractable lipid in the plant part isabout 3%, about 4%, about 5%, about 6% or about 7%, and

ii) extracting lipid from the plant part, wherein the extracted lipidhas the following features in the total fatty acid content of the lipid;

i) the level of DHA is about 3%, about 4%, about 5%, about 6% or about7%,

ii) the level of palmitic acid is between about 2% and about 16%,

iii) the level of myristic acid is less than about 2%,

iv) the level of oleic acid is between about 30% and about 60%,preferably between about 45% and about 60%,

v) the level of LA is between about 4% and about 20%,

vi) the level of ALA is between about 2% and about 16%,

vii) the level of GLA is less than about 4%,

viii) the level of SDA is less than about 6%, or less than about 4%,

ix) the level of ETA is less than about 6%, or less than about 4%,

x) the level of ETrA less than about 1%,

xi) the level of EPA is less than about 10% and/or the level of EPA is0.5-2.0 fold the level of DHA,

xii) the level of DPA is less than about 4%,

xiii) the level of total saturated fatty acids in the total fatty acidcontent of the extracted lipid is between about 4% and about 25%,

xiv) the level of total monounsaturated fatty acids in the total fattyacid content of the extracted lipid is between about 30% and about 70%,

xv) the level of total polyunsaturated fatty acids in the total fattyacid content of the extracted lipid is between about 15% and about 75%,preferably between about 15% and about 30%,

xvi) the level of new ω6 fatty acids in the total fatty acid content ofthe extracted lipid is between about 0.5% and about 10%,

xvii) the level of total ω3 fatty acids in the total fatty acid contentof the extracted lipid is between about 10% and about 20%,

xviii) the level of new ω3 fatty acids in the total fatty acid contentof the extracted lipid is between about 3% and about 20%,

xix) the ratio of total ω6 fatty acids:total ω3 fatty acids in the fattyacid content of the extracted lipid is between about 0.05 and about 3.0,preferably less than about 0.50,

xx) the ratio of new ω6 fatty acids:new ω3 fatty acids in the fatty acidcontent of the extracted lipid is between about 0.03 and about 3.0,

xxi) the triacylglycerol (TAG) content of the lipid is at least about70%, and xxii) the lipid is essentially free of cholesterol. In anembodiment, the lipid comprises tri-DHA TAG (TAG 66:18). Morepreferably, the lipid is essentially free of SDA and ETA, and/or has notbeen treated with a transesterification process after it was extractedfrom the plant or plant part.

Also provided is a process for producing extracted plant lipid,comprising the steps of

i) obtaining a plant part comprising lipid, the lipid comprising fattyacids in an esterified form, the fatty acids comprising oleic acid,palmitic acid, ω6 fatty acids which comprise linoleic acid (LA), ω3fatty acids which comprise α-linolenic acid (ALA) and docosahexaenoicacid (DHA), and one or more of stearidonic acid (SDA), eicosapentaenoicacid (EPA), docosapentaenoic acid (DPA) and eicosatetraenoic acid (ETA),wherein (i) the level of DHA in the total fatty acid content of theextracted lipid is between 7% and 20%, (ii) the level of palmitic acidin the total fatty acid content of the extracted lipid is between 2% and16%, (iii) the level of myristic acid (C14:0) in the total fatty acidcontent of the extracted lipid is less than 6%, (iv) the level of oleicacid in the total fatty acid content of the extracted lipid is between1% and 30% or between 30% and 60%, (v) the level of linoleic acid (LA)in the total fatty acid content of the extracted lipid is between 4% and35%, (vi) the level of α-linolenic acid (ALA) in the total fatty acidcontent of the extracted lipid is between 4% and 40%, (vii) the level ofeicosatrienoic acid (ETrA) in the total fatty acid content of theextracted lipid is less than 4%, (viii) the level of total saturatedfatty acids in the total fatty acid content of the extracted lipid isbetween 4% and 25%, (ix) the ratio of total ω6 fatty acids:total ω3fatty acids in the fatty acid content of the extracted lipid is between1.0 and 3.0 or between 0.1 and 1, (x) the triacylglycerol (TAG) contentof the lipid is at least 70%, and (xi) at least 70% of the DHAesterified in the form of TAG is in the sn-1 or sn-3 position of theTAG. %, and

ii) extracting lipid from the plant part,

wherein the level of DHA in the total fatty acid content of theextracted lipid is about 7% to 20%.

Also provided is lipid, or oil comprising the lipid, produced using aprocess of the invention.

In another aspect, the present invention provides a process forproducing ethyl esters of polyunsaturated fatty acids, the processcomprising transesterifying triacylglycerols in extracted plant lipid,wherein the extracted plant lipid comprises fatty acids esterified inthe form, the fatty acids comprising oleic acid, palmitic acid, ω6 fattyacids which comprise linoleic acid (LA), ω3 fatty acids which compriseα-linolenic acid (ALA), and docosahexaenoic acid (DHA), and optionallyone or more of stearidonic acid (SDA), eicosapentaenoic acid (EPA),docosapentaenoic acid (DPA) and eicosatetraenoic acid (ETA), wherein thelevel of DHA in the total fatty acid content of the extracted lipid isabout 7% to 20%, thereby producing the ethyl esters.

In a preferred embodiment, the extracted lipid has one or more of thefeatures defined above.

In another aspect, the present invention provides a process forproducing ethyl esters of polyunsaturated fatty acids, the processcomprising transesterifying triacylglycerols in extracted plant lipid,wherein the extracted plant lipid comprises fatty acids esterified inthe form of the triacylglycerols, the fatty acids comprising oleic acid,palmitic acid, ω6 fatty acids which comprise linoleic acid (LA), ω3fatty acids which comprise α-linolenic acid (ALA) and docosahexaenoicacid (DHA), and one or more of stearidonic acid (SDA), eicosapentaenoicacid (EPA), docosapentaenoic acid (DPA) and eicosatetraenoic acid (ETA),wherein (i) the level of DHA in the total fatty acid content of theextracted lipid is about 3%, about 4%, about 5%, about 6% or between 7%and 20%, (ii) the level of palmitic acid in the total fatty acid contentof the extracted lipid is between 2% and 16%, (iii) the level ofmyristic acid (C14:0) in the total fatty acid content of the extractedlipid is less than 6%, (iv) the level of oleic acid in the total fattyacid content of the extracted lipid is between 1% and 30% or between 30%and 60%, (v) the level of linoleic acid (LA) in the total fatty acidcontent of the extracted lipid is between 4% and 35%, (vi) the level ofα-linolenic acid (ALA) in the total fatty acid content of the extractedlipid is between 4% and 40%, (vii) the level of eicosatrienoic acid(ETrA) in the total fatty acid content of the extracted lipid is lessthan 4%, (viii) the level of total saturated fatty acids in the totalfatty acid content of the extracted lipid is between 4% and 25%, (ix)the ratio of total ω6 fatty acids:total ω3 fatty acids in the fatty acidcontent of the extracted lipid is between 1.0 and 3.0 or between 0.1 and1, (x) the triacylglycerol (TAG) content of the lipid is at least 70%,and (xi) at least 70% of the DHA esterified in the form of TAG is in thesn-1 or sn-3 position of the TAG, thereby producing the ethyl esters. Inan embodiment, the extracted plant lipid has one or more or all of thefollowing features

-   -   i) the level of palmitic acid in the total fatty acid content of        the extracted lipid is between 2% and 15%,    -   ii) the level of myristic acid (C14:0) in the total fatty acid        content of the extracted lipid is less than 1%,    -   xxxv) the level of oleic acid in the total fatty acid content of        the extracted lipid is between about 3% and about 30%, between        about 6% and about 30%, between 1% and about 20%, between about        45% and about 60%, or is about 30%,    -   xxxvi) the level of linoleic acid (LA) in the total fatty acid        content of the extracted lipid is between about 4% and about        20%, or between about 4% and 17%,    -   xxxvii) the level of α-linolenic acid (ALA) in the total fatty        acid content of the extracted lipid is between about 7% and        about 40%, between about 10% and about 35%, between about 20%        and about 35%, or between about 4% and 16%,    -   xxxviii) the level of γ-linolenic acid (GLA) in the total fatty        acid content of the extracted lipid is less than 4%, less than        about 3%, less than about 2%, less than about 1%, less than        about 0.5%, between 0.05% and 7%, between 0.05% and 4%, or        between 0.05% and about 3%, or between 0.05% and about 2%,    -   xxxix) the level of stearidonic acid (SDA) in the total fatty        acid content of the extracted lipid is less than about 4%, less        than about 3%, between about 0.05% and about 7%, between about        0.05% and about 4%, between about 0.05% and about 3%, or between        0.05% and about 2%,    -   xl) the level of eicosatetraenoic acid (ETA) in the total fatty        acid content of the extracted lipid is less than about 4%, less        than about 1%, less than about 0.5%, between about 0.05% and        about 5%, between about 0.05% and about 4%, between about 0.05%        and about 3%, or between about 0.05% and about 2%,    -   xli) the level of eicosatrienoic acid (ETrA) in the total fatty        acid content of the extracted lipid is less than about 2%, less        than about 1%, between 0.05% and 4%, between 0.05% and 3%, or        between 0.05% and about 2%, or between 0.05% and about 1%,    -   xlii) the level of eicosapentaenoic acid (EPA) in the total        fatty acid content of the extracted lipid is less than 4%, less        than about 3%, less than about 2%, between 0.05% and 10%,        between 0.05% and 5%, or between 0.05% and about 3%, or between        0.05% and about 2%,    -   xliii) the level of docosapentaenoic acid (DPA) in the total        fatty acid content of the extracted lipid is less than 4%, less        than about 3%, less than about 2%, between 0.05% and 8%, between        0.05% and 5%, or between 0.05% and about 3%, or between 0.05%        and about 2%,    -   xliv) the level of DHA in the total fatty acid content of the        extracted lipid is about 8%, about 9%, about 10%, about 11%,        about 12%, about 13%, about 14%, about 15%, about 16%, about        17%, about 18%, between about 8% and 20%, between about 10% and        20%, between about 11% and 20%, between about 10% and about 16%,        or between about 14% and 20%,    -   xlv) the lipid comprises ω6-docosapentaenoic acid        (22:5^(Δ4,7,10,13,16)) in its fatty acid content,    -   xlvi) the lipid is essentially free of ω6-docosapentaenoic acid        (22:5^(Δ4,7,10,13,16)) in its fatty acid content,    -   xlvii) the lipid is essentially free of SDA, EPA and ETA in its        fatty acid content,    -   xlviii) the level of total saturated fatty acids in the total        fatty acid content of the extracted lipid is between about 4%        and about 20%, or between about 6% and about 20%,    -   xlix) the level of total monounsaturated fatty acids in the        total fatty acid content of the extracted lipid is between about        4% and about 35%, between about 8% and about 25%, or between 8%        and about 22%,    -   l) the level of total polyunsaturated fatty acids in the total        fatty acid content of the extracted lipid is between about 20%        and about 75%, between about 50% and about 75%, or between about        60% and about 75%,    -   li) the level of total ω6 fatty acids in the total fatty acid        content of the extracted lipid is between about 35% and about        50%, between about 20% and about 35%, between about 6% and 20%,        less than 20%, less than about 16%, less than about 10%, between        about 1% and about 16%, between about 2% and about 10%, or        between about 4% and about 10%,    -   lii) the level of new ω6 fatty acids in the total fatty acid        content of the extracted lipid is less than about 10%, less than        about 8%, less than about 6%, less than 4%, between about 1% and        about 20%, between about 1% and about 10%, between about 0.5%        and about 8%, or between about 0.5% and 4%,    -   liii) the level of total ω3 fatty acids in the total fatty acid        content of the extracted lipid is between 36% and about 65%,        between 40% and about 60%, between about 20% and about 35%,        between about 10% and about 20%, about 25%, about 30%, about 35%        or about 40%,    -   liv) the level of new ω3 fatty acids in the total fatty acid        content of the extracted lipid is between 9% and about 33%,        between about 10% and about 20%, between about 20% and about        30%, between about 12% and about 25%, about 13%, about 15%,        about 17% or about 20%,    -   lv) the ratio of total ω6 fatty acids:total ω3 fatty acids in        the fatty acid content of the extracted lipid is between about        0.1 and about 0.5, less than about 0.50, less than about 0.40,        less than about 0.30, less than about 0.20, less than about        0.15, about 1.0, about 0.1 or about 0.2,    -   lvi) the ratio of new ω6 fatty acids:new ω3 fatty acids in the        fatty acid content of the extracted lipid is between about 1.0        and about 3.0, between about 0.1 and about 1, between about 0.1        and about 0.5, less than about 0.50, less than about 0.40, less        than about 0.30, less than about 0.20, less than about 0.15,        about 0.1, about 0.2 or about 1.0,    -   lvii) the fatty acid composition of the lipid is based on an        efficiency of conversion of oleic acid to DHA of at least about        10%, at least about 15%, at least about 20%, between about 10%        and about 50%, between about 10% and about 30%, or between about        10% and about 25%,    -   lviii) the fatty acid composition of the lipid is based on an        efficiency of conversion of LA to DHA of at least about 15%, at        least about 20%, at least about 22%, at least about 25%, between        about 15% and about 50%, between about 20% and about 40%, or        between about 20% and about 30%,    -   lix) the fatty acid composition of the lipid is based on an        efficiency of conversion of ALA to DHA of at least about 17%, at        least about 22%, at least about 24%, between about 17% and about        55%, between about 22% and about 35%, or between about 24% and        about 35%,    -   lx) the total fatty acid in the extracted lipid has less than 1%        C20:1,    -   lxi) the triacylglycerol (TAG) content of the lipid is at least        about 80%, at least about 90%, at least 95%, between about 70%        and about 99%, or between about 90% and about 99%,    -   lxii) the lipid comprises diacylglycerol (DAG),    -   lxiii) the lipid comprises less than about 10%, less than about        5%, less than about 1%, or between about 0.001% and about 5%,        free (non-esterified) fatty acids and/or phospholipid, or is        essentially free thereof,    -   lxiv) at least 80%, of the DHA esterified in the form of TAG is        in the sn-1 or sn-3 position of the TAG,    -   lxv) the most abundant DHA-containing TAG species in the lipid        is DHA/18:3/18:3 (TAG 58:12), and    -   lxvi) the lipid comprises tri-DHA TAG (TAG 66:18).

With specific regard to the above aspect, in an embodiment one or moreor all of the following apply

i) the lipid is in the form of an oil, wherein the oil comprises one ormore sterols such as one or more or all of campesterol, Δ5-stigmasterol,eburicol, β-sitosterol, Δ5-avenasterol, Δ7-stigmasterol andΔ7-avenasterol, and optionally the oil comprises less than 10 mg ofsterols/g of oil and/or the oil is essentially free of cholesterol,

ii) the lipid is in the form of an oil from an oilseed such as oilseedis a Brassica sp oilseed or canola seed,

iii) the level of DHA in the total fatty acid content of the extractedplant lipid is about 3%, about 4%, about 5%, about 6%, or is between 7%and 20%.

In a further aspect, the present invention provides a chimeric geneticconstruct comprising in order a first gene, a second gene, a third gene,a fourth gene, a fifth gene and a sixth gene which are all covalentlylinked on a single DNA molecule,

wherein the first, second and third genes are joined together as a firstgene cluster and the fourth, fifth and sixth genes are joined togetheras a second gene cluster,wherein each gene comprises a promoter, a coding region and atranscription terminator and/or polyadenylation region such that eachpromoter is operably linked to the coding region and transcriptionterminator and/or polyadenylation region,wherein each promoter is independently identical or different to theother promoters such that the DNA molecule comprises three, four, fiveor six different promoters,wherein one or more or all of the promoters are heterologous withrespect to the coding region to which it is operably linked,wherein the direction of transcription of the first gene is away fromthe third gene and opposite to the direction of transcription of thethird gene,wherein the direction of transcription of the fourth gene is away fromthe sixth gene and opposite to the direction of transcription of thesixth gene,wherein the direction of transcription of the second gene is the same asfor the first gene or the third gene,wherein the direction of transcription of the fifth gene is the same asfor the fourth gene or the sixth gene,wherein the transcription terminator and/or polyadenylation region ofthe second gene is spaced apart from the promoter of the first or thirdgenes, whichever is closer, by a first spacer region of between about0.2 and about 3.0 kilobases,wherein the first gene cluster is spaced apart from the second genecluster by a second spacer region of between about 1.0 and about 10.0kilobases, andwherein the transcription terminator and/or polyadenylation region ofthe fifth gene is spaced apart from the promoter of the fourth or sixthgenes, whichever is closer, by a third spacer region of between about0.2 and about 3.0 kilobases.

In an embodiment, the DNA molecule comprises a seventh gene which isspaced apart from the first gene cluster or the second gene cluster,whichever is closer, by a spacer region of between about 1.0 and about10.0 kilobases.

In another embodiment, the DNA molecule comprises two or more differenttranscription terminator and/or polyadenylation regions.

In yet a further embodiment, at least one of the spacer regionscomprises a matrix attachment region (MAR).

In a further embodiment, the DNA molecule comprises right and leftborder regions flanking the genes and is a T-DNA molecule.

In another embodiment, the genetic construct is in an Agrobacterium cellor is integrated into the genome of a plant cell.

In a preferred embodiment, at least one of the genes encodes a fattyacid desaturase or a fatty acid elongase.

In another embodiment, the genetic construct comprises genes encoding aset of enzymes as defined herein, and/or wherein one or more of thegenes encode an enzyme as defined herein.

In a further aspect, the present invention provides an isolated and/orexogenous polynucleotide comprising:

i) a sequence of nucleotides selected from any one of SEQ ID NOs: 1 to9, 11, 14, 18, 22, 23, 28, 34, 35, 39 or 45, and/or

ii) a sequence of nucleotides which are at least 95% identical or 99%identical to one or more of the sequences set forth in SEQ ID NOs: 1 to9, 11, 14, 18, 22, 23, 28, 34, 35, 39 or 45.

In a particularly preferred embodiment, the isolated and/or exogenouspolynucleotide comprises:

i) a sequence of nucleotides of SEQ ID NO: 2, and/or

ii) a sequence of nucleotides which are at least 95% identical or 99%identical to the sequence set forth in SEQ ID NO: 2.

In another aspect, the present invention provides a vector or geneticconstruct comprising the polynucleotide of the invention and/or thegenetic construct of the invention.

In an embodiment, the sequence of nucleotides selected from any one ofSEQ ID NOs: 11, 14, 18, 22, 23, 28, 34, 35, 39 or 45, or the sequence ofnucleotides which is at least 95% identical or 99% identical to one ormore of the sequences set forth in SEQ ID NOs: 11, 14, 18, 22, 23, 28,34, 35, 39 or 45, is operably linked to a promoter.

In a further aspect, the present invention provides a host cellcomprising exogenous polynucleotides encoding one of the following setsof enzymes;

i) an ω3-desaturase, a Δ6-desaturase, a Δ5-desaturase, a Δ4-desaturase,a Δ6-elongase and a Δ5-elongase,

ii) a Δ15-desaturase, a Δ6-desaturase, a Δ5-desaturase, a Δ4-desaturase,a Δ6-elongase and a Δ5-elongase,

iii) a Δ12-desaturase, a Δ6-desaturase, a Δ5-desaturase, aΔ4-desaturase, a Δ6-elongase and an Δ5-elongase,

iv) a Δ12-desaturase, a ω3-desaturase or a Δ15-desaturase, aΔ6-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ6-elongase and anΔ5-elongase,

v) an ω3-desaturase, a Δ8-desaturase, a Δ5-desaturase, a Δ4-desaturase,a Δ9-elongase and an Δ5-elongase,

vi) a Δ15-desaturase, a Δ8-desaturase, a Δ5-desaturase, a Δ4-desaturase,a Δ9-elongase and a Δ5-elongase,

vii) a Δ12-desaturase, a Δ8-desaturase, a Δ5-desaturase, aΔ4-desaturase, a Δ9-elongase and an Δ5-elongase, or

viii) a Δ12-desaturase, a ω3-desaturase or a Δ15-desaturase, aΔ8-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ9-elongase and anΔ5-elongase, and wherein each polynucleotide is operably linked to oneor more promoters that are capable of directing expression of saidpolynucleotides in the cell.

In an embodiment, the cell comprises lipid as defined above, or whereinone or more or all of the desaturases or elongases have one or more ofthe features as defined above.

In another aspect, the present invention provides a host cell comprising

i) a first exogenous polynucleotide encoding a Δ12-desaturase whichcomprises amino acids having a sequence as provided in SEQ ID NO:10, abiologically active fragment thereof, or an amino acid sequence which isat least 50% identical to SEQ ID NO:10, and

ii) a second exogenous polynucleotide encoding a ω3-desaturase whichcomprises amino acids having a sequence as provided in SEQ ID NO: 12, abiologically active fragment thereof, or an amino acid sequence which isat least 50% identical to SEQ ID NO:12,

wherein each polynucleotide is operably linked to one or more promotersthat are capable of directing expression of said polynucleotides in thecell.

In a further aspect, the present invention provides a host cellcomprising one or more of the polynucleotide of the invention, thegenetic construct of the invention, or the vector or genetic constructof the invention.

In an embodiment, the cell is in a plant, in a plant part and/or is amature plant seed cell.

In an embodiment, the plant or plant seed is an oilseed plant or anoilseed, respectively.

Also provided is a transgenic non-human organism comprising a cell ofthe invention. Preferably, the transgenic non-human organism is atransgenic plant, preferably an oilseed plant or Arabidopsis thaliana.In an embodiment, the plant is a Brassica plant, preferably B. napus orB. juncea, or a plant other than Arabidopsis thaliana.

In another aspect, the present invention provides an oilseed plantcomprising

a) lipid in its seed, the lipid comprising fatty acids in an esterifiedform, and

b) exogenous polynucleotides encoding one of the following sets ofenzymes;

-   -   i) a Δ12-desaturase, a fungal ω3-desaturase and/or fungal        Δ15-desaturase, a Δ6-desaturase, a Δ5-desaturase, a        Δ4-desaturase, a Δ6-elongase and an Δ5-elongase, or    -   ii) a Δ12-desaturase, a fungal ω3-desaturase and/or fungal        Δ15-desaturase, a Δ8-desaturase, a Δ5-desaturase, a        Δ4-desaturase, a Δ9-elongase and an Δ5-elongase,

wherein each polynucleotide is operably linked to one or moreseed-specific promoters that are capable of directing expression of saidpolynucleotides in developing seed of the plant, wherein the fatty acidscomprise oleic acid, palmitic acid, ω6 fatty acids which compriselinoleic acid (LA) and γ-linolenic acid (GLA), ω3 fatty acids whichcomprise α-linolenic acid (ALA), stearidonic acid (SDA),docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA), andoptionally eicosapentaenoic acid (EPA) and/or eicosatetraenoic acid(ETA), and wherein the level of DHA in the total fatty acid content ofthe lipid is about 7% to 20%.

Examples of oilseed plants include, but are not limited to, Brassicasp., Gossypium hirsutum, Linum usitatissimum, Helianthus sp., Carthamustinctorius, Glycine max, Zea mays, Arabidopsis thaliana, Sorghumbicolor, Sorghum vulgare, Avena sativa, Trifolium sp., Elaesisguineenis, Nicotiana benthamiana, Hordeum vulgare, Lupinusangustifolius, Oryza sativa, Oryza glaberrima, Camelina sativa, orCrambe abyssinica. In an embodiment, the oilseed plant is a canola,Glycine max, Camelina sativa or Arabidopsis thaliana plant. In analternate embodiment, the oilseed plant is other than A. thaliana.

In an embodiment, one or more of the desaturases is capable of using anacyl-CoA substrate. In a preferred embodiment, one or more of theΔ6-desaturase, Δ5-desaturase, Δ4-desaturase and Δ8-desaturase, ifpresent, is capable of using an acyl-CoA substrate, preferably each ofthe i) Δ6-desaturase, Δ5-desaturase and Δ4-desaturase or ii)Δ5-desaturase, Δ4-desaturase and Δ8-desaturase is capable of using anacyl-CoA substrate. In an embodiment, a Δ12-desaturase and/or anω3-desaturase is capable of using an acyl-CoA substrate. The acyl-CoAsubstrate is preferably an ALA-CoA, ETA-CoA, DPA-CoA, ETrA-CoA, LA-CoA,GLA-CoA, or ARA-CoA.

In an embodiment, mature, harvested seed of the plant has a DHA contentof at least about 28 mg per gram seed, preferably at least about 32 mgper gram seed, at least about 36 mg per gram seed, at least about 40 mgper gram seed, more preferably at least about 44 mg per gram seed or atleast about 48 mg per gram seed. The maximum DHA content may be about 80to about 100 mg per gram seed, or about 80 mg or about 100 mg per gramseed.

In a further aspect, the present invention provides a Brassica napus, B.juncea or Camelina sativa plant which is capable of producing seedcomprising DHA, wherein mature, harvested seed of the plant has a DHAcontent of at least about 28 mg per gram seed, preferably at least about32 mg per gram seed, at least about 36 mg per gram seed, at least about40 mg per gram seed, more preferably at least about 44 mg per gram seedor at least about 48 mg per gram seed. The maximum DHA content may beabout 80 to about 100 mg per gram seed, or about 80 mg or about 100 mgper gram seed.

In another aspect, the present invention provides plant cell of a plantof the invention comprising the exogenous polynucleotides.

Also provided is a plant part, preferably a seed, which has one or moreof the following features

i) is from a plant of the invention,

ii) comprises lipid as defined herein,

iii) can be used in a process of the invention,

iv) comprises a genetic construct of the invention, or

v) comprises a set of exogenous polynucleotides as defined herein.

In yet another aspect, the present invention provides mature, harvestedBrassica napus, B. juncea or Camelina sativa seed comprising DHA and amoisture content of between about 4% and about 15% by weight, whereinthe DHA content of the seed at least about 28 mg per gram seed,preferably at least about 32 mg per gram seed, at least about 36 mg pergram seed, at least about 40 mg per gram seed, more preferably at leastabout 44 mg per gram seed or at least about 48 mg per gram seed. Themaximum DHA content may be about 80 to about 100 mg per gram seed, orabout 80 mg or about 100 mg per gram seed.

In an embodiment, the cell of the invention, the transgenic organism ofthe invention, the oilseed plant of the invention, the Brassica napus,B. juncea or Camelina sativa plant of the invention, the plant part ofthe invention, or the seed of the invention, which can be used toproduce extracted lipid comprising one or more or all of the featuresdefined herein.

In yet a further aspect, the present invention provides a method ofproducing a cell of the invention, the method comprising

a) introducing into the cell, preferably a cell which is not capable ofsynthesising a LC-PUFA, the gene construct of the invention, theisolated and/or exogenous polynucleotide of the invention, the vector orgenetic construct of the invention, one or more of the combinations ofexogenous polynucleotides defined herein,

b) optionally, expressing the genes or polynucleotide(s) in the cell;

c) optionally, analysing the fatty acid composition of the cell, and

d) optionally, selecting a cell which express the genes orpolynucleotide(s).

In an embodiment, the lipid in the cell has one or more of the featuresdefined herein.

In another embodiment, the gene construct, the isolated and/or exogenouspolynucleotide, the vector, the genetic construct or combinations ofexogenous polynucleotides, become stably integrated into the genome ofthe cell.

In a further embodiment, the cell is a plant cell, and the methodfurther comprises the step of regenerating a transformed plant from thecell of step a).

In another embodiment, the genes and/or exogenous polynucleotide(s) areexpressed transiently in the cell.

Also provided is a cell produced using a method of the invention.

In another aspect, the present invention provides a method of producingseed, the method comprising,

a) growing a plant of the invention, or a plant which produces a part asdefined herein, preferably in a field as part of a population of atleast 1000 such plants or in an area of at least 1 hectare planted at astandard planting density,

b) harvesting seed from the plant or plants, and

c) optionally, extracting lipid from the seed, preferably to produce oilwith a total DHA yield of at least 60 kg DHA/hectare.

In an embodiment, the plant, plant cell, plant part or seed of theinvention has one or more of the following features

-   -   i) the oil is as defined herein,    -   ii) the plant part or seed is capable of being used in a process        of the invention,    -   iii) the exogenous polynucleotides are comprised in a genetic        construct of the invention,    -   iv) the exogenous polynucleotides comprise an exogenous        polynucleotide of the invention,    -   v) the plant cell is a cell of the invention, and    -   vi) the seed was produced according to the method of the        invention.

In another aspect, the present invention provides a method of producingone or more fatty acid desaturases and/or fatty acid elongases, or oneor more fatty acid desaturases and one or more fatty acid elongases, themethod comprising expressing in a cell or cell free expression systemthe gene construct of the invention, the isolated and/or exogenouspolynucleotide of the invention, the vector or genetic construct of theinvention, one or more of the combinations of exogenous polynucleotidesdefined herein, preferably in a developing oilseed in an oilseed plantin the field.

In a further aspect, the present invention provides lipid, or oil,produced by, or obtained from, using the process of the invention, thecell of the invention, the transgenic organism of the invention, theoilseed plant of the invention, the Brassica napus, B. juncea orCamelina sativa plant of the invention, the plant part of the invention,the seed of the invention, or the plant, plant cell, plant part or seedof the invention.

In an embodiment, the lipid or oil is obtained by extraction of oil froman oilseed. Examples of oil from oilseeds include, but are not limitedto, canola oil (Brassica napus, Brassica rapa ssp.), mustard oil(Brassica juncea), other Brassica oil, sunflower oil (Helianthus annus),linseed oil (Linum usitatissimum), soybean oil (Glycine max), saffloweroil (Carthamus tinctorius), corn oil (Zea mays), tobacco oil (Nicotianatabacum), peanut oil (Arachis hypogaea), palm oil, cottonseed oil(Gossypium hirsutum), coconut oil (Cocos nucifera), avocado oil (Perseaamericana), olive oil (Olea europaea), cashew oil (Anacardiumoccidentale), macadamia oil (Macadamia intergrifolia), almond oil(Prunus amygdalus) or Arabidopsis seed oil (Arabidopsis thaliana).

In a further aspect, the present invention provides fatty acid producedby, or obtained from, using the process of the invention, the cell ofthe invention, the transgenic organism of the invention, the oilseedplant of the invention, the Brassica napus, B. juncea or Camelina sativaplant of the invention, the plant part of the invention, the seed of theinvention, or the plant, plant cell, plant part or seed of theinvention. Preferably the fatty acid is DHA. The fatty acid may be in amixture of fatty acids having a fatty acid composition as describedherein. In an embodiment, the fatty acid is non-esterified.

Also provided is seedmeal obtained from seed of the invention. Preferredseedmeal includes, but not necessarily limited to, Brassica napus, B.juncea, Camelina sativa or Glycine max seedmeal. In an embodiment, theseedmeal comprises an exogenous polynucleotide(s) and/or geneticconstructs as defined herein.

In another aspect, the present invention provides a compositioncomprising one or more of a lipid or oil of the invention, the fattyacid of the invention, the genetic construct of the invention, theisolated and/or exogenous polynucleotide of the invention, the vector orgenetic construct of the invention, the cell according of the invention,the transgenic organism of the invention, the oilseed plant of theinvention, the Brassica napus, B. juncea or Camelina sativa plant of theinvention, the plant part of the invention, the seed of the invention,the plant, plant cell, plant part or seed of the invention, or theseedmeal of the invention. In embodiments, the composition comprises acarrier suitable for pharmaceutical, food or agricultural use, a seedtreatment compound, a fertiliser, another food or feed ingredient, oradded protein or vitamins.

Also provided is feedstuffs, cosmetics or chemicals comprising one ormore of the lipid or oil of the invention, the fatty acid of theinvention, the genetic construct of the invention, the isolated and/orexogenous polynucleotide of the invention, the vector or geneticconstruct of the invention, the cell according of the invention, thetransgenic organism of the invention, the oilseed plant of theinvention, the Brassica napus, B. juncea or Camelina sativa plant of theinvention, the plant part of the invention, the seed of the invention,the plant, plant cell, plant part or seed of the invention, the seedmealof the invention, or the composition of the invention.

In another aspect, the present invention provides a method of producinga feedstuff, the method comprising mixing one or more of the lipid oroil of the invention, the fatty acid of the invention, the geneticconstruct of the invention, the isolated and/or exogenous polynucleotideof the invention, the vector or genetic construct of the invention, thecell according of the invention, the transgenic organism of theinvention, the oilseed plant of the invention, the Brassica napus, B.juncea or Camelina sativa plant of the invention, the plant part of theinvention, the seed of the invention, the plant, plant cell, plant partor seed of the invention, the seedmeal of the invention, or thecomposition of the invention, with at least one other food ingredient.

In another aspect, the present invention provides a method of treatingor preventing a condition which would benefit from a PUFA, the methodcomprising administering to a subject one or more of the lipid or oil ofthe invention, the fatty acid of the invention, the genetic construct ofthe invention, the isolated and/or exogenous polynucleotide of theinvention, the vector or genetic construct of the invention, the cellaccording of the invention, the transgenic organism of the invention,the oilseed plant of the invention, the Brassica napus, B. juncea orCamelina sativa plant of the invention, the plant part of the invention,the seed of the invention, the plant, plant cell, plant part or seed ofthe invention, the seedmeal of the invention, the composition of theinvention, or the feedstuff of the invention.

Examples of conditions which would benefit from a PUFA include, but arenot limited to, cardiac arrhythmia's, angioplasty, inflammation, asthma,psoriasis, osteoporosis, kidney stones, AIDS, multiple sclerosis,rheumatoid arthritis, Crohn's disease, schizophrenia, cancer, foetalalcohol syndrome, attention deficient hyperactivity disorder, cysticfibrosis, phenylketonuria, unipolar depression, aggressive hostility,adrenoleukodystophy, coronary heart disease, hypertension, diabetes,obesity, Alzheimer's disease, chronic obstructive pulmonary disease,ulcerative colitis, restenosis after angioplasty, eczema, high bloodpressure, platelet aggregation, gastrointestinal bleeding,endometriosis, premenstrual syndrome, myalgic encephalomyelitis, chronicfatigue after viral infections or an ocular disease.

Also provided is the use of one or more of the lipid or oil of theinvention, the fatty acid of the invention, the genetic construct of theinvention, the isolated and/or exogenous polynucleotide of theinvention, the vector or genetic construct of the invention, the cellaccording of the invention, the transgenic organism of the invention,the oilseed plant of the invention, the Brassica napus, B. juncea orCamelina sativa plant of the invention, the plant part of the invention,the seed of the invention, the plant, plant cell, plant part or seed ofthe invention, the seedmeal of the invention, the composition of theinvention, or the feedstuff of the invention for the manufacture of amedicament for treating or preventing a condition which would benefitfrom a PUFA. The production of the medicament may comprise mixing theoil of the invention with a pharmaceutically acceptable carrier, fortreatment of a condition as described herein. The method may comprisefirstly purifying the oil and/or transesterification, and/orfractionation of the oil to increase the level of DHA. In a particularembodiment, the method comprises treating the lipid or oil such ascanola oil to convert the fatty acids in the oil to alkyl esters such asmethyl or ethyl esters. Further treatment such as fractionation ordistillation may be applied to enrich the lipid or oil for the DHA. In apreferred embodiment, the medicament comprises ethyl esters of DHA. Inan even more preferred embodiment, the level of ethyl esters of DHA inthe medicament is between 30% and 50%. The medicament may furthercomprise ethyl esters of EPA, such as between 30% and 50% of the totalfatty acid content in the medicament. Such medicaments are suitable foradministration to human or animal subjects for treatment of medicalconditions as described herein.

In another aspect, the present invention provides a method of tradingseed, comprising obtaining seed of the invention, and trading theobtained seed for pecuniary gain.

In an embodiment, obtaining the seed comprises cultivating plants of theinvention and/or harvesting the seed from the plants.

In another embodiment, obtaining the seed further comprises placing theseed in a container and/or storing the seed.

In a further embodiment, obtaining the seed further comprisestransporting the seed to a different location.

In yet another embodiment, the method further comprises transporting theseed to a different location after the seed is traded.

In a further embodiment, the trading is conducted using electronic meanssuch as a computer.

In yet a further aspect, the present invention provides a process ofproducing bins of seed comprising:

a) swathing, windrowing and/or or reaping above-ground parts of plantscomprising seed of the invention,

b) threshing and/or winnowing the parts of the plants to separate theseed from the remainder of the plant parts, and

c) sifting and/or sorting the seed separated in step b), and loading thesifted and/or sorted seed into bins, thereby producing bins of seed.

In an embodiment, where relevant, the lipid or oil, preferably seedoil,of, or useful for, the invention has fatty levels about those providedin a Table in the Examples section, such as seed 14 of Table 16.

Any embodiment herein shall be taken to apply mutatis mutandis to anyother embodiment unless specifically stated otherwise.

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended for the purpose ofexemplification only. Functionally-equivalent products, compositions andmethods are clearly within the scope of the invention, as describedherein.

Throughout this specification, unless specifically stated otherwise orthe context requires otherwise, reference to a single step, compositionof matter, group of steps or group of compositions of matter shall betaken to encompass one and a plurality (i.e. one or more) of thosesteps, compositions of matter, groups of steps or group of compositionsof matter.

The invention is hereinafter described by way of the followingnon-limiting Examples and with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. 1. Aerobic DHA biosynthesis pathways.

FIG. 2. Map of the T-DNA insertion region between the left and rightborders of pJP3416-GA7. RB denotes right border; LB, left border; TER,transcription terminator/polyadenylation region; PRO, promoter; Codingregions are indicated above the arrows, promoters and terminators belowthe arrows. Micpu-Δ6D, Micromonas pusilla Δ6-desaturase; Pyrco-Δ6E,Pyramimonas cordata Δ6-elongase; Pavsa-Δ5D, Pavlova salinaΔ5-desaturase; Picpa-ω3D, Pichia pastoris ω3-desaturase; Pavsa-Δ4D, P.salina Δ4-desaturase; Lackl-Δ12D, Lachancea kluyveri Δ12-desaturase;Pyrco-Δ5E, Pyramimonas cordata Δ5-elongase. NOS denotes theAgrobacterium tumefaciens nopaline synthase transcriptionterminator/polyadenylation region; FP1, Brassica napus truncated napinpromoter; FAE1, Arabidopsis thaliana FAE1 promoter; Lectin, Glycine maxlectin transcription terminator/polyadenylation region; Cnl1 and Cnl2denotes the Linum usitatissimum conlinin1 or conlinin2 promoter orterminator. MAR denotes the Rb7 matrix attachment region from Nicotianatabacum.

FIG. 3. Map of the T-DNA insertion region between the left and rightborders of pJP3404. Labels are as in FIG. 2.

FIG. 4. Map of the insertion region between the left and right bordersof pJP3367. Labels are as in FIG. 2.

FIG. 5. DHA levels as a percentage of total fatty acids in seed lipidfrom multiple independent transgenic Arabidopsis thaliana seeds in boththe T₂ and T₃ generations. The bracketed T₂ events were taken to T₃.Events from both the Columbia and fad2 mutant A. thaliana backgroundsare shown.

FIG. 6. Oil content (w/w) vs. DHA content, as a percentage of totalfatty acid content of lipid from transgenic Arabidopsis thaliana seeds.

FIG. 7. Representative RT-PCR gel showing the low expression of theΔ6-desaturase gene relative to the other transgenes in the T-DNA of B.napus embryos transformed using pJP3416-GA7. Lanes from the left showRT-PCR products: 1, DNA size markers; lane 2, Δ12 desaturase; lane 3,ω3-desaturase; lane 4, Δ6-desaturase (low expression); lane 5,Δ6-elongase; lane 6, Δ5-desaturase; lane 7, Δ5-elongase; lane 8,Δ4-desaturase.

FIG. 8. Percentage of ALA plotted against percentage of oleic acid, eachas a percentage of total fatty acids in lipid obtained from transgenic35S:LEC2 Brassica napus somatic embryos.

FIG. 9. Positional distribution analysis by NMR on A) Tuna oil and, B)transgenic DHA Arabidopsis seed oil. The peaks labelled ‘DHA-alpha’represent the amount of DHA present at the sn-1 and sn-3 positions ofTAG (with no positional preference this would equal 66% of total DHA)whilst the peaks labelled ‘DHA-beta’ represent the amount of DHA presentat the sn-2 position of TAG (with no preference this would equal 33% ofDHA).

FIG. 10. LC-MS analysis of major DHA-containing triacylglycerol speciesin transgenic A. thaliana developing (grey) and mature (black) seeds.The number following the DHA denotes the total number of carbon atomsand total number of double bonds in the other two fatty acids. ThereforeDHA/34:1 can also be designated TAG 56:7, etc.

FIG. 11. Map of the T-DNA insertion region between the left and rightborders of pORE04+1ABGBEC_Cowpea_EPA_insert. Labels are as in FIG. 2;SSU, Arabidopsis thaliana rubisco small subunit promoter.

FIG. 12. Map of the binary vector pJP3364 showing the NotI restrictionsite into which the candidate Δ12-desaturases were cloned.

FIG. 13. BoxPlot generated using SigmaPlot showing the percentage offatty acid 20:4ω6 (ARA) in seed lipid of Arabidopsis T2 seed populationstransformed with pFN045-pFN050. The boundary of each box closest to zeroindicates the 25th percentile, a line within each box marks the median,and the boundary of each box farthest from zero indicates the 75thpercentile. Error bars shown above and below each box indicate the 90thand 10th percentiles.

FIG. 14. Average level of ARA as a percentage of the total fatty acidcontent in seed lipid of Arabidopsis T2 seed transformed withpFN045-pFN050.

FIG. 15. BoxPlot showing the percentage of fatty acid 20:2ω6 (EDA) inseed lipid of Arabidopsis T2 seed populations transformed withpFN045-pFN050. The BoxPlot represents values as described in FIG. 13.

FIG. 16. BoxPlot showing the percentage of ARA in seed lipid ofArabidopsis T4 seed populations transformed with pFN045-pFN050. TheBoxPlot represents values as described in FIG. 13.

FIG. 17. Average level of ARA as a percentage of the total fatty acidcontent in seed lipid of Arabidopsis T4 seed populations transformedwith pFN045-pFN050.

FIG. 18. BoxPlot showing the percentage of EDA in seed lipid ofArabidopsis T4 seed populations transformed with pFN045-pFN050. TheBoxPlot represents values as described in FIG. 13.

FIG. 19. (A) Basic phytosterol structure with ring and side chainnumbering. (B) Chemical structures of some of the phytosterols.

FIG. 20. Phylogenetic tree of known LPAATs.

FIG. 21. The various acyl exchange enzymes which transfer fatty acidsbetween PC, CoA pools, and TAG pools. Adapted from Singh et al. (2005).

KEY TO THE SEQUENCE LISTING

SEQ ID NO: 1—pJP3416-GA7 nucleotide sequence.SEQ ID NO:2—pGA7-mod_B nucleotide sequence.SEQ ID NO:3—pGA7-mod_C nucleotide sequence.SEQ ID NO:4—pGA7-mod_D nucleotide sequence.SEQ ID NO:5—pGA7-mod_E nucleotide sequence.SEQ ID NO:6—pGA7-mod_F nucleotide sequence.SEQ ID NO:7—pGA7-mod_G nucleotide sequence.SEQ ID NO:8—pORE04+11ABGBEC_Cowpea_EPA_insert nucleotide sequence.SEQ ID NO:9—Codon-optimized open reading frame for expression ofLachancea kluyveri Δ12 desaturase in plants.SEQ ID NO: 10—Lachancea kluyveri Δ12-desaturase.SEQ ID NO:11—Codon-optimized open reading frame for expression of Pichiapastoris ω3 desaturase in plants.SEQ ID NO: 12—Pichia pastoris ω3 desaturase.SEQ ID NO: 13—Open reading frame encoding Micromonas pusillaΔ6-desaturase.SEQ ID NO:14—Codon-optimized open reading frame for expression ofMicromonas pusilla Δ6-desaturase in plants (version 1).SEQ ID NO:15—Codon-optimized open reading frame for expression ofMicromonas pusilla Δ6-desaturase in plants (version 2).SEQ ID NO: 16—Micromonas pusilla Δ6-desaturase.SEQ ID NO:17—Open reading frame encoding Ostreococcus lucimarinusΔ6-desaturase.SEQ ID NO: 18—Codon-optimized open reading frame for expression ofOstreococcus lucimarinus Δ6-desaturase in plants.SEQ ID NO: 19—Ostreococcus lucimarinus Δ6-desaturase.SEQ ID NO:20—Ostreococcus tauri Δ6-desaturase.SEQ ID NO:21—Open reading frame encoding Pyramimonas cordataΔ6-elongase.SEQ ID NO:22—Codon-optimized open reading frame for expression ofPyramimonas cordata Δ6-elongase in plants (truncated at 3′ end andencoding functional elongase) (version 1).SEQ ID NO:23—Codon-optimized open reading frame for expression ofPyramimonas cordata Δ6-elongase in plants (truncated at 3′ end andencoding functional elongase) (version 2).SEQ ID NO:24—Codon-optimized open reading frame for expression ofPyramimonas cordata Δ6-elongase in plants (truncated at 3′ end andencoding functional elongase) (version 3).SEQ ID NO:25—Pyramimonas cordata Δ6-elongase.SEQ ID NO:26—Truncated Pyramimonas cordata Δ6-elongase.SEQ ID NO:27—Open reading frame encoding Pavlova salina Δ5-desaturase.SEQ ID NO:28—Codon-optimized open reading frame for expression ofPavlova salina Δ5-desaturase in plants (version 1).SEQ ID NO:29—Codon-optimized open reading frame for expression ofPavlova salina Δ5-desaturase in plants (version 2).SEQ ID NO:30—Pavlova salina Δ5-desaturase.SEQ ID NO:31—Open reading frame encoding Pyramimonas cordataΔ5-desaturase.SEQ ID NO:32—Pyramimonas cordata Δ5-desaturase.SEQ ID NO:33—Open reading frame encoding Pyramimonas cordataΔ5-elongase.SEQ ID NO:34—Codon-optimized open reading frame for expression ofPyramimonas cordata Δ5-elongase in plants (version 1).SEQ ID NO:35—Codon-optimized open reading frame for expression ofPyramimonas cordata Δ5-elongase in plants (version 2).SEQ ID NO:36—Codon-optimized open reading frame for expression ofPyramimonas cordata Δ5-elongase in plants (version 3).SEQ ID NO:37—Pyramimonas cordata Δ5-elongase.SEQ ID NO:38—Open reading frame encoding Pavlova salina Δ4-desaturase.SEQ ID NO:39—Codon-optimized open reading frame for expression ofPavlova salina Δ4-desaturase in plants (version 1).SEQ ID NO:40—Codon-optimized open reading frame for expression ofPavlova salina Δ4-desaturase in plants (version 2).SEQ ID NO:41—Pavlova salina Δ4-desaturase.SEQ ID NO:42—Open reading frame encoding Isochrysis galbana Δ9-elongase.SEQ ID NO:43—Isochrysis galbana Δ9-elongase.SEQ ID NO:44—Open reading frame encoding Emiliania huxleyi CCMP1516Δ9-elongase.SEQ ID NO:45—Codon-optimized open reading frame for expression ofEmiliania huxleyi Δ9-elongase in plants.SEQ ID NO:46—Emiliania huxleyi CCMP1516 Δ9-elongase.SEQ ID NO:47—Open reading frame encoding Pavlova pinguis Δ9-elongase.SEQ ID NO:48—Pavlova pinguis Δ9-elongase.SEQ ID NO:49—Open reading frame encoding Pavlova salina Δ9-elongase.SEQ ID NO:50—Pavlova salina Δ9-elongase.SEQ ID NO:51—Open reading frame encoding Pavlova salina Δ8-desaturase.SEQ ID NO:52—Pavlova salina Δ8-desaturase.SEQ ID NO:53—P19 viral suppressor.SEQ ID NO:54—V2 viral suppressor.SEQ ID NO:55—P38 viral suppressor.SEQ ID NO:56—Pe-P0 viral suppressor.SEQ ID NO:57—RPV-P0 viral suppressor.SEQ ID NO:58—Open reading frame encoding P19 viral suppressor.SEQ ID NO:59—Open reading frame encoding V2 viral suppressor.SEQ ID NO:60—Open reading frame encoding P38 viral suppressor.SEQ ID NO:61—Open reading frame encoding Pe-P0 viral suppressor.SEQ ID NO:62—Open reading frame encoding RPV-P0 viral suppressor.SEQ ID NO: 63—Arabidopsis thaliana LPAAT2.SEQ ID NO: 64—Limnanthes alba LPAAT.SEQ ID NO: 65—Saccharomyces cerevisiae LPAAT.SEQ ID NO: 66—Micromonas pusilla LPAAT.SEQ ID NO: 67—Mortierella alpina LPAAT.SEQ ID NO: 68—Braccisa napus LPAAT.SEQ ID NO: 69—Brassica napus LPAAT.SEQ ID NO: 70—Phytophthora infestans ω3 desaturase.SEQ ID NO: 71—Thalassiosira pseudonana ω3 desaturase.SEQ ID NO: 72—Pythium irregulare ω3 desaturase.

DETAILED DESCRIPTION OF THE INVENTION General Techniques and Definitions

Unless specifically defined otherwise, all technical and scientificterms used herein shall be taken to have the same meaning as commonlyunderstood by one of ordinary skill in the art (e.g., in cell culture,molecular genetics, fatty acid synthesis, transgenic plants, proteinchemistry, and biochemistry).

Unless otherwise indicated, the recombinant protein, cell culture, andimmunological techniques utilized in the present invention are standardprocedures, well known to those skilled in the art. Such techniques aredescribed and explained throughout the literature in sources such as, J.Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons(1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbour Laboratory Press (1989), T. A. Brown (editor), EssentialMolecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press(1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A PracticalApproach, Volumes 1-4, IRL Press (1995 and 1996), F. M. Ausubel et al.(editors), Current Protocols in Molecular Biology, Greene Pub.Associates and Wiley-Interscience (1988, including all updates untilpresent), Ed Harlow and David Lane (editors), Antibodies: A LaboratoryManual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al.(editors), Current Protocols in Immunology, John Wiley & Sons (includingall updates until present).

The term “and/or”, e.g., “X and/or Y” shall be understood to mean either“X and Y” or “X or Y” and shall be taken to provide explicit support forboth meanings or for either meaning.

As used herein, the term “about”, unless stated to the contrary, refersto +/−10%, more preferably +/−5%, more preferably +/−1% of thedesignated value.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

Selected Definitions

As used herein, the terms “extracted plant lipid” and “isolated plantlipid” refer to a lipid composition which has been extracted from, forexample by crushing, a plant or part thereof such as seed. The extractedlipid can be a relatively crude composition obtained by, for example,crushing a plant seed, or a more purified composition where most, if notall, of one or more or each of the water, nucleic acids, proteins andcarbohydrates derived from the plant material have been removed.Examples of purification methods are described below. In an embodiment,the extracted or isolated plant lipid comprises at least about 60%, atleast about 70%, at least about 80%, at least about 90%, or at leastabout 95% (w/w) lipid by weight of the composition. The lipid may besolid or liquid at room temperature, when liquid it is considered to bean oil. In an embodiment, extracted lipid of the invention has not beenblended with another lipid such as DHA not produced by another source(for example, DHA from fish oil). In an embodiment, following extractionthe ratio of one or more or all of, oleic acid to DHA, palmitic acid toDHA, linoleic acid to DHA, and total ω6 fatty acids:total ω3 fattyacids, has not been significantly altered (for example, no greater thana 10% or 5% alteration) when compared to the ratio in the intact seed orcell. In an another embodiment, the extracted plant lipid has not beenexposed to a procedure, such as hydrogenation or fractionation, whichmay alter the ratio of one or more or all of, oleic acid to DHA,palmitic acid to DHA, linoleic acid to DHA, and total ω6 fatty acids:total ω3 fatty acids, when compared to the ratio in the intact seed orcell. When the extracted plant lipid of the invention is comprised in anoil, the oil may further comprise non-fatty acid molecules such assterols.

As used herein, the terms “extracted plant oil” and “isolated plant oil”refer to a substance or composition comprising extracted plant lipid orisolated plant lipid and which is a liquid at room temperature. The oilis obtained from a plant or part thereof such as seed. The extracted orisolated oil can be a relatively crude composition obtained by, forexample, crushing a plant seed, or a more purified composition wheremost, if not all, of one or more or each of the water, nucleic acids,proteins and carbohydrates derived from the plant material have beenremoved. The composition may comprise other components which may belipid or non-lipid. In an embodiment, the oil composition comprises atleast about 60%, at least about 70%, at least about 80%, at least about90%, or at least about 95% (w/w) extracted plant lipid. In anembodiment, extracted oil of the invention has not been blended withanother oil such as DHA not produced by another source (for example, DHAfrom fish oil). In an embodiment, following extraction, the ratio of oneor more or all of, oleic acid to DHA, palmitic acid to DHA, linoleicacid to DHA, and total ω6 fatty acids:total ω3 fatty acids, has not beensignificantly altered (for example, no greater than a 10% or 5%alteration) when compared to the ratio in the intact seed or cell. In ananother embodiment, the extracted plant oil has not been exposed to aprocedure, such as hydrogenation or fractionation, which may alter theratio of one or more or all of, oleic acid to DHA, palmitic acid to DHA,linoleic acid to DHA, and total ω6 fatty acids: total ω3 fatty acids,when compared to the ratio in the intact seed or cell. Extracted plantoil of the invention may comprise non-fatty acid molecules such assterols.

As used herein, an “oil” is a composition comprising predominantly lipidand which is a liquid at room temperature. For instance, oil of theinvention preferably comprises at least 75%, at least 80%, at least 85%or at least 90% lipid by weight. Typically, a purified oil comprises atleast 90% triacylglycerols (TAG) by weight of the lipid in the oil.Minor components of an oil such as diacylglycerols (DAG), free fattyacids (FFA), phospholipid and sterols may be present as describedherein.

As used herein, the term “fatty acid” refers to a carboxylic acid (ororganic acid), often with a long aliphatic tail, either saturated orunsaturated. Typically fatty acids have a carbon-carbon bonded chain ofat least 8 carbon atoms in length, more preferably at least 12 carbonsin length. Most naturally occurring fatty acids have an even number ofcarbon atoms because their biosynthesis involves acetate which has twocarbon atoms. The fatty acids may be in a free state (non-esterified) orin an esterified form such as part of a triglyceride, diacylglyceride,monoacylglyceride, acyl-CoA (thio-ester) bound or other bound form. Thefatty acid may be esterified as a phospholipid such as aphosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,phosphatidylglycerol, phosphatidylinositol or diphosphatidylglycerolforms.

“Saturated fatty acids” do not contain any double bonds or otherfunctional groups along the chain. The term “saturated” refers tohydrogen, in that all carbons (apart from the carboxylic acid [—COOH]group) contain as many hydrogens as possible. In other words, the omega(ω) end contains 3 hydrogens (CH3-) and each carbon within the chaincontains 2 hydrogens (—CH2-).

“Unsaturated fatty acids” are of similar form to saturated fatty acids,except that one or more alkene functional groups exist along the chain,with each alkene substituting a singly-bonded “—CH2-CH2-” part of thechain with a doubly-bonded “—CH═CH—” portion (that is, a carbon doublebonded to another carbon). The two next carbon atoms in the chain thatare bound to either side of the double bond can occur in a cis or transconfiguration.

As used herein, the term “monounsaturated fatty acid” refers to a fattyacid which comprises at least 12 carbon atoms in its carbon chain andonly one alkene group (carbon-carbon double bond) in the chain. As usedherein, the terms “polyunsaturated fatty acid” or “PUFA” refer to afatty acid which comprises at least 12 carbon atoms in its carbon chainand at least two alkene groups (carbon-carbon double bonds).

As used herein, the terms “long-chain polyunsaturated fatty acid” and“LC-PUFA” refer to a fatty acid which comprises at least 20 carbon atomsin its carbon chain and at least two carbon-carbon double bonds, andhence include VLC-PUFAs. As used herein, the terms “very long-chainpolyunsaturated fatty acid” and “VLC-PUFA” refer to a fatty acid whichcomprises at least 22 carbon atoms in its carbon chain and at leastthree carbon-carbon double bonds. Ordinarily, the number of carbon atomsin the carbon chain of the fatty acids refers to an unbranched carbonchain. If the carbon chain is branched, the number of carbon atomsexcludes those in sidegroups. In one embodiment, the long-chainpolyunsaturated fatty acid is an ω3 fatty acid, that is, having adesaturation (carbon-carbon double bond) in the third carbon-carbon bondfrom the methyl end of the fatty acid. In another embodiment, thelong-chain polyunsaturated fatty acid is an ω6 fatty acid, that is,having a desaturation (carbon-carbon double bond) in the sixthcarbon-carbon bond from the methyl end of the fatty acid. In a furtherembodiment, the long-chain polyunsaturated fatty acid is selected fromthe group consisting of; arachidonic acid (ARA, 20:4Δ5,8,11,14; ω6),eicosatetraenoic acid (ETA, 20:4Δ8,11,14,17, ω3), eicosapentaenoic acid(EPA, 20:5Δ5,8,11,14,17; ω3), docosapentaenoic acid (DPA,22:5Δ7,10,13,16,19, ω3), or docosahexaenoic acid (DHA,22:6Δ4,7,10,13,16,19, ω3). The LC-PUFA may also be dihomo-γ-linoleicacid (DGLA) or eicosatrienoic acid (ETrA, 20:3Δ11,14,17, ω3). It wouldreadily be apparent that the LC-PUFA that is produced according to theinvention may be a mixture of any or all of the above and may includeother LC-PUFA or derivatives of any of these LC-PUFA. In a preferredembodiment, the ω3 fatty acids are at least DHA, preferably, DPA andDHA, or EPA, DPA and DHA.

Furthermore, as used herein the terms “long-chain polyunsaturated fattyacid” and “very long-chain polyunsaturated fatty acid” refer to thefatty acid being in a free state (non-esterified) or in an esterifiedform such as part of a triglyceride, diacylglyceride, monoacylglyceride,acyl-CoA bound or other bound form. The fatty acid may be esterified asa phospholipid such as a phosphatidylcholine (PC),phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol,phosphatidylinositol or diphosphatidylglycerol forms. Thus, the LC-PUFAmay be present as a mixture of forms in the lipid of a cell or apurified oil or lipid extracted from cells, tissues or organisms. Inpreferred embodiments, the invention provides oil comprising at least75% or at least 85% triacylglycerols, with the remainder present asother forms of lipid such as those mentioned, with at least saidtriacylglycerols comprising the LC-PUFA. The oil may subsequently befurther purified or treated, for example by hydrolysis with a strongbase to release the free fatty acids, or by distillation or the like.

As used herein, “total ω6 fatty acids” or “total ω6 fatty acid content”or the like refers to the sum of all the ω6 fatty acids, esterified andnon-esterified, in the extracted lipid, oil, recombinant cell, plantpart or seed, as the context determines, expressed as a percentage ofthe total fatty acid content. These ω6 fatty acids include (if present)LA, GLA, DGLA, ARA, EDA and ω6-DPA, and exclude any ω3 fatty acids andmonounsaturated fatty acids.

As used herein, “new ω6 fatty acids” or “new ω6 fatty acid content” orthe like refers to the sum of all the ω6 fatty acids excluding LA,esterified and non-esterified, in the extracted lipid, oil, recombinantcell, plant part or seed, as the context determines, expressed as apercentage of the total fatty acid content. These new ω6 fatty acids arethe fatty acids that are produced in the cells, plants, plant parts andseeds of the invention by the expression of the genetic constructs(exogenous polynucleotides) introduced into the cells, and include (ifpresent) GLA, DGLA, ARA, EDA and ω6-DPA, but exclude LA and any ω3 fattyacids and monounsaturated fatty acids. Exemplary total ω6 fatty acidcontents and new ω6 fatty acid contents are determined by conversion offatty acids in a sample to FAME and analysis by GC, as described inExample 1.

As used herein, “total ω3 fatty acids” or “total ω3 fatty acid content”or the like refers to the sum of all the ω3 fatty acids, esterified andnon-esterified, in the extracted lipid, oil, recombinant cell, plantpart or seed, as the context determines, expressed as a percentage ofthe total fatty acid content. These ω3 fatty acids include (if present)ALA, SDA, ETrA, ETA, EPA, DPA and DHA, and exclude any ω6 fatty acidsand monounsaturated fatty acids.

As used herein, “new ω3 fatty acids” or “new ω3 fatty acid content” orthe like refers to the sum of all the ω3 fatty acids excluding ALA,esterified and non-esterified, in the extracted lipid, oil, recombinantcell, plant part or seed, as the context determines, expressed as apercentage of the total fatty acid content. These new ω3 fatty acids arethe fatty acids that are produced in the cells, plants, plant parts andseeds of the invention by the expression of the genetic constructs(exogenous polynucleotides) introduced into the cells, and include (ifpresent) SDA, ETrA, ETA, EPA, DPA and DHA, but exclude ALA and any ω6fatty acids and monounsaturated fatty acids. Exemplary total ω3 fattyacid contents and new ω3 fatty acid contents are determined byconversion of fatty acids in a sample to FAME and analysis by GC, asdescribed in Example 1.

The desaturase, elongase and acyl transferase proteins and genesencoding them that may be used in the invention are any of those knownin the art or homologues or derivatives thereof. Examples of such genesand encoded protein sizes are listed in Table 1. The desaturase enzymesthat have been shown to participate in LC-PUFA biosynthesis all belongto the group of so-called “front-end” desaturases.

As used herein, the term “front-end desaturase” refers to a member of aclass of enzymes that introduce a double bond between the carboxyl groupand a pre-existing unsaturated part of the acyl chain of lipids, whichare characterized structurally by the presence of an N-terminalcytochrome b5 domain, along with a typical fatty acid desaturase domainthat includes three highly conserved histidine boxes (Napier et al.,1997).

Activity of any of the elongases or desaturases for use in the inventionmay be tested by expressing a gene encoding the enzyme in a cell suchas, for example, a yeast cell, a plant cell or preferably in somaticembryos or transgenic plants, and determining whether the cell, embryoor plant has an increased capacity to produce LC-PUFA compared to acomparable cell, embryo or plant in which the enzyme is not expressed.

In one embodiment one or more of the desaturases and/or elongases foruse in the invention can purified from a microalga, i.e. is identical inamino acid sequence to a polypeptide which can be purified from amicroalga.

Whilst certain enzymes are specifically described herein as“bifunctional”, the absence of such a term does not necessarily implythat a particular enzyme does not possess an activity other than thatspecifically defined.

Desaturases

As used herein, the term “desaturase” refers to an enzyme which iscapable of introducing a carbon-carbon double bond into the acyl groupof a fatty acid substrate which is typically in an esterified form suchas, for example, acyl-CoA esters. The acyl group may be esterified to aphospholipid such as phosphatidylcholine (PC), or to acyl carrierprotein (ACP), or in a preferred embodiment to CoA. Desaturasesgenerally may be categorized into three groups accordingly. In oneembodiment, the desaturase is a front-end desaturase.

As used herein, a “Δ4-desaturase” refers to a protein which performs adesaturase reaction that introduces a carbon-carbon double bond at the4^(th) carbon-carbon bond from the carboxyl end of a fatty acidsubstrate. The “Δ4-desaturase” is at least capable of converting DPA toDHA. The desaturation step to produce DHA from DPA is catalysed by aΔ4-desaturase in organisms other than mammals, and a gene encoding thisenzyme has been isolated from the freshwater protist species Euglenagracilis and the marine species Thraustochytrium sp. (Qiu et al., 2001;Meyer et al., 2003). In one embodiment, the Δ4-desaturase comprisesamino acids having a sequence as provided in SEQ ID NO:41, or aThraustochytrium sp. Δ4-desaturase, a biologically active fragmentthereof, or an amino acid sequence which is at least 80% identical toSEQ ID NO:41.

TABLE 1 Cloned genes involved in LC-PUFA biosynthesis Type of AccessionProtein size Enzyme organism Species Nos. (aa's) ReferencesΔ4-desaturase Protist Euglena gracilis AY278558 541 Meyer et al., 2003Algae Pavlova lutherii AY332747 445 Tonon et al, 2003 Isochrysis galbanaAAV33631 433 Pereira et al., 2004b Pavlova salina AAY15136 447 Zhou etal., 2007 Thraustochytrid Thraustochytrium aureum AAN75707 515 N/AAAN75708 AAN75709 AAN75710 Thraustochytrium sp. AAM09688 519 Qiu et al.2001 ATCC21685 Δ5-desaturase Mammals Homo sapiens AF199596 444 Cho etal., 1999b Leonard et al., 2000b Nematode Caenorhabditis elegansAF11440, 447 Michaelson et al., 1998b; NM_069350 Watts and Browse, 1999bFungi Mortierella alpina AF067654 446 Michaelson et al., 1998a; Knutzonet al., 1998 Pythium irregulare AF419297 456 Hong et al., 2002aDictyostelium discoideum AB022097 467 Saito et al., 2000 Saprolegniadiclina 470 WO02081668 Diatom Phaeodactylum tricornutum AY082392 469Domergue et al., 2002 Algae Thraustochytrium sp AF489588 439 Qiu et al.,2001 Thraustochytrium aureum 439 WO02081668 Isochrysis galbana 442WO02081668 Moss Marchantia polymorpha AY583465 484 Kajikawa et al., 2004Δ6-desaturase Mammals Homo sapiens NM_013402 444 Cho et al., 1999a;Leonard et al., 2000 Mus musculus NM_019699 444 Cho et al., 1999aNematode Caenorhabditis elegans Z70271 443 Napier et al., 1998 PlantsBorago officinales U79010 448 Sayanova et al., 1997 Echium AY055117Garcia-Maroto et al., 2002 AY055118 Primula vialii AY234127 453 Sayanovaet al., 2003 Anemone leveillei AF536525 446 Whitney et al., 2003 MossesCeratodon purpureus AJ250735 520 Sperling et al., 2000 Marchantiapolymorpha AY583463 481 Kajikawa et al., 2004 Physcomitrella patensCAA11033 525 Girke et al., 1998 Fungi Mortierella alpina AF110510 457Huang et al., 1999; AB020032 Sakuradani et al., 1999 Pythium irregulareAF419296 459 Hong et al., 2002a Mucor circinelloides AB052086 467 NCBI*Rhizopus sp. AY320288 458 Zhang et al., 2004 Saprolegnia diclina 453WO02081668 Diatom Phaeodactylum tricornutum AY082393 477 Domergue etal., 2002 Bacteria Synechocystis L11421 359 Reddy et al., 1993 AlgaeThraustochytrium aureum 456 WO02081668 Bifunctional Δ5/ Fish Danio rerioAF309556 444 Hastings et al., 2001 Δ6-desaturase C20 Algae Euglenagracilis AF139720 419 Wallis and Browse, 1999 Δ8-desaturase PlantsBorago officinales AAG43277 446 Sperling et al., 2001 Δ6-elongaseNematode Caenorhabditis elegans NM_069288 288 Beaudoin et al., 2000Mosses Physcomitrella patens AF428243 290 Zank et al., 2002 Marchantiapolymorpha AY583464 290 Kajikawa et al., 2004 Fungi Mortierella alpinaAF206662 318 Parker-Barnes et al., 2000 Algae Pavlova lutheri** 501 WO03078639 Thraustochytrium AX951565 271 WO 03093482 Thraustochytrium sp**AX214454 271 WO 0159128 PUFA-elongase Mammals Homo sapiens AF231981 299Leonard et al., 2000b; Leonard et al., 2002 Rattus norvegicus AB071985299 Inagaki et al., 2002 Rattus norvegicus** AB071986 267 Inagaki etal., 2002 Mus musculus AF170907 279 Tvrdik et al., 2000 Mus musculusAF170908 292 Tvrdik et al., 2000 Fish Danio rerio AF532782 291 (282)Agaba et al., 2004 Danio rerio** NM_199532 266 Lo et al., 2003 WormCaenorhabditis elegans Z68749 309 Abbott et al., 1998 Beaudoin et al.,2000 Algae Thraustochytrium aureum** AX464802 272 WO 0208401-A2 Pavlovalutheri** 320 WO 03078639 Δ9-elongase Algae Isochrysis galbana AF390174263 Qi et al., 2002 Euglena gracilis 258 WO 08/128241 Δ5-elongase AlgaeOstreococcus tauri AAV67798 300 Meyer et al., 2004 Pyramimonas cordata268 WO 2010/057246 Pavlova sp. CCMP459 AAV33630 277 Pereira et al.,2004b Pavlova salina AAY15135 302 Robert et al., 2009 DiatomThalassiosira pseudonana AAV67800 358 Meyer et al., 2004 FishOncorhynchus mykiss CAM55862 295 WO 06/008099 Moss Marchantia polymorphaBAE71129 348 Kajikawa et al., 2006 *http://www.ncbi.nlm.nih.gov/**Function not proven/not demonstrated

As used herein, a “Δ5-desaturase” refers to a protein which performs adesaturase reaction that introduces a carbon-carbon double bond at the5^(th) carbon-carbon bond from the carboxyl end of a fatty acidsubstrate. Examples of Δ5-desaturases are listed in Ruiz-Lopez et al.(2012) and Petrie et al. (2010a) and in Table 1 herein. In oneembodiment, the Δ5-desaturase comprises amino acids having a sequence asprovided in SEQ ID NO:30, a biologically active fragment thereof, or anamino acid sequence which is at least 80% identical to SEQ ID NO:30. Inanother embodiment, the Δ5-desaturase comprises amino acids having asequence as provided in SEQ ID NO:32, a biologically active fragmentthereof, or an amino acid sequence which is at least 53% identical toSEQ ID NO:32. In another embodiment, the Δ5-desaturase is fromThraustochytrium sp or Emiliania huxleyi.

As used herein, a “Δ6-desaturase” refers to a protein which performs adesaturase reaction that introduces a carbon-carbon double bond at the6^(th) carbon-carbon bond from the carboxyl end of a fatty acidsubstrate. Examples of Δ6-desaturases are listed in Ruiz-Lopez et al.(2012) and Petrie et al. (2010a) and in Table 1 herein. PreferredΔ6-desaturases are from Micromonas pusilla, Pythium irregulare orOstreococcus taurii.

In an embodiment, the Δ6-desaturase is further characterised by havingat least two, preferably all three and preferably in a plant cell, ofthe following: i) greater Δ6-desaturase activity on t-linolenic acid(ALA, 18:3Δ9,12,15, ω3) than linoleic acid (LA, 18:2Δ9,12, ω6) as fattyacid substrate; ii) greater Δ6-desaturase activity on ALA-CoA as fattyacid substrate than on ALA joined to the sn-2 position of PC as fattyacid substrate; and iii) Δ8-desaturase activity on ETrA. Examples ofsuch Δ6-desaturases are provided in Table 2.

In an embodiment the Δ6-desaturase has greater activity on an ω3substrate than the corresponding ω6 substrate and has activity on ALA toproduce octadecatetraenoic acid (stearidonic acid, SDA, 18:4Δ6,9,12, 15,ω3) with an efficiency of at least 30%, more preferably at least 40%, ormost preferably at least 50% when expressed from an exogenouspolynucleotide in a recombinant cell such as a plant cell, or at least35% when expressed in a yeast cell. In one embodiment, the Δ6-desaturasehas greater activity, for example, at least about a 2-fold greaterΔ6-desaturase activity, on ALA than LA as fatty acid substrate. Inanother embodiment, the Δ6-desaturase has greater activity, for example,at least about 5 fold greater Δ6-desaturase activity or at least 10-foldgreater activity, on ALA-CoA as fatty acid substrate than on ALA joinedto the sn-2 position of PC as fatty acid substrate. In a furtherembodiment, the Δ6-desaturase has activity on both fatty acid substratesALA-CoA and on ALA joined to the sn-2 position of PC.

TABLE 2 Desaturases demonstrated to have activity on an acyl-CoAsubstrate Type of Accession Protein size Enzyme organism Species Nos.(aa's) References Δ6-desaturase Algae Mantoniella squamata CAQ30479 449Hoffmann et al., 2008 Ostreococcus tauri AAW70159 456 Domergue et al.,2005 Micromonas pusilla EEH58637 Petrie et al., 2010a (SEQ ID NO: 13)Δ5-desaturase Algae Mantoniella squamata CAQ30478 482 Hoffmann et al.,2008 Plant Anemone leveillei N/A Sayanova et al., 2007 ω3-desaturaseFungi Pythium aphanidermatum FW362186.1 359 Xue et al., 2012;WO2008/054565 Fungi Phytophthora sojae FW362214.1 363 Xue et al., 2012;(oomycete) WO2008/054565 Fungi Phytophthora ramorum FW362213.1 361 Xueet al., 2012; (oomycete) WO2008/054565

In one embodiment, the Δ6-desaturase has no detectable Δ5-desaturaseactivity on ETA. In another embodiment, the Δ6-desaturase comprisesamino acids having a sequence as provided in SEQ ID NO:16, SEQ ID NO:19or SEQ ID NO:20, a biologically active fragment thereof, or an aminoacid sequence which is at least 77% identical to SEQ ID NO:16, SEQ IDNO:19 or SEQ ID NO:20. In another embodiment, the Δ6-desaturasecomprises amino acids having a sequence as provided in SEQ ID NO:19 orSEQ ID NO:20, a biologically active fragment thereof, or an amino acidsequence which is at least 67% identical to one or both of SEQ ID NO:19or SEQ ID NO:20. The Δ6-desaturase may also have Δ8-desaturase activity.

As used herein, a “Δ8-desaturase” refers to a protein which performs adesaturase reaction that introduces a carbon-carbon double bond at the8^(th) carbon-carbon bond from the carboxyl end of a fatty acidsubstrate. The Δ8-desaturase is at least capable of converting ETrA toETA. Examples of Δ8-desaturases are listed in Table 1. In oneembodiment, the Δ8-desaturase comprises amino acids having a sequence asprovided in SEQ ID NO:52, a biologically active fragment thereof, or anamino acid sequence which is at least 80% identical to SEQ ID NO:52.

As used herein, an “ω3-desaturase” refers to a protein which performs adesaturase reaction that introduces a carbon-carbon double bond at the3rd carbon-carbon bond from the methyl end of a fatty acid substrate. Aω3-desaturase therefore may convert LA to ALA and GLA to SDA (all C18fatty acids), or DGLA to ETA and/or ARA to EPA (C20 fatty acids). Someω3-desaturases (group I) have activity only on C18 substrates, such asplant and cyanobacterial ω3-desaturases. Such ω3-desaturases are alsoΔ15-desaturases. Other ω3-desaturases have activity on C20 substrateswith no activity (group II) or some activity (group III) on C18substrates. Such ω3-desaturases are also Δ17-desaturases. Preferredω3-desaturases are group III type which convert LA to ALA, GLA to SDA,DGLA to ETA and ARA to EPA, such as the Pichia pastoris ω3-desaturase(SEQ ID NO: 12). Examples of ω3-desaturases include those described byPereira et al. (2004a) (Saprolegnia diclina ω3-desaturase, group II),Horiguchi et al. (1998), Berberich et al. (1998) and Spychalla et al.(1997) (C. elegans ω3-desaturase, group III). In a preferred embodiment,the ω3-desaturase is a fungal ω3-desaturase. As used herein, a “fungalω3-desaturase” refers to an ω3-desaturase which is from a fungal source,including an oomycete source, or a variant thereof whose amino acidsequence is at least 95% identical thereto. Genes encoding numerousω3-desaturases have been isolated from fungal sources such as, forexample, from Phytophthora infestans (Accession No. CAJ30870,WO2005083053; SEQ ID NO: 70), Saprolegnia diclina (Accession No.AAR20444, Pereira et al., 2004a & U.S. Pat. No. 7,211,656), Pythiumirregulare (WO2008022963, Group II; SEQ ID NO: 72), Mortierella alpina(Sakuradani et al., 2005; Accession No. BAD91495; WO2006019192),Thalassiosira pseudonana (Armbrust et al., 2004; Accession No.XP_002291057; WO2005012316, SEQ ID NO: 71), Lachancea kluyveri (alsoknown as Saccharomyces kluyveri; Oura et al., 2004; Accession No.AB118663). Xue et al. (2012) describes ω3-desaturases from the oomycetesPythium aphanidermatum, Phytophthora sojae, and Phytophthora ramorumwhich were able to efficiently convert ω6 fatty acid substrates to thecorresponding ω3 fatty acids, with a preference for C20 substrates, i.e.they had stronger Δ17-desaturase activity than Δ15-desaturase activity.These enzymes lacked Δ12-desaturase activity, but could use fatty acidsin both acyl-CoA and phospholipid fraction as substrates.

In a more preferred embodiment, the fungal ω3-desaturase is the Pichiapastoris (also known as Komagataella pastoris)ω3-desaturase/Δ15-desaturase (Zhang et al., 2008; Accession No.EF116884; SEQ ID NO: 12), or a polypeptide which is at least 95%identical thereto.

In an embodiment, the ω3-desaturase is at least capable of convertingone of ARA to EPA, DGLA to ETA, GLA to SDA, both ARA to EPA and DGLA toETA, both ARA to EPA and GLA to SDA, or all three of these.

In one embodiment, the ω3-desaturase has Δ17-desaturase activity on aC20 fatty acid which has at least three carbon-carbon double bonds,preferably ARA. In another embodiment, the ω3-desaturase hasΔ15-desaturase activity on a C18 fatty acid which has threecarbon-carbon double bonds, preferably GLA. Preferably, both activitiesare present.

As used herein, a “Δ12-desaturase” refers to a protein which performs adesaturase reaction that introduces a carbon-carbon double bond at the12^(th) carbon-carbon bond from the carboxyl end of a fatty acidsubstrate. Δ12-desaturases typically convert eitheroleoyl-phosphatidylcholine or oleoyl-CoA tolinoleoyl-phosphatidylcholine (18:1-PC) or linoleoyl-CoA (18:1-CoA),respectively. The subclass using the PC linked substrate are referred toas phospholipid-dependent Δ12-desaturases, the latter subclass asacyl-CoA dependent Δ12-desaturases. Plant and fungal Δ12-desaturases aregenerally of the former sub-class, whereas animal Δ12-desaturases are ofthe latter subclass, for example the Δ12-desaturases encoded by genescloned from insects by Zhou et al. (2008). Many other Δ12-desaturasesequences can be easily identified by searching sequence databases.

As used herein, a “Δ15-desaturase” refers to a protein which performs adesaturase reaction that introduces a carbon-carbon double bond at the15^(th) carbon-carbon bond from the carboxyl end of a fatty acidsubstrate. Numerous genes encoding 25 Δ15-desaturases have been clonedfrom plant and fungal species. For example, U.S. Pat. No. 5,952,544describes nucleic acids encoding plant Δ15-desaturases (FAD3). Theseenzymes comprise amino acid motifs that were characteristic of plantΔ15-desaturases. WO200114538 describes a gene encoding soybean FAD3.Many other Δ15-desaturase sequences can be easily identified bysearching sequence databases.

As used herein, a “Δ17-desaturase” refers to a protein which performs adesaturase reaction that introduces a carbon-carbon double bond at the17^(th) carbon-carbon bond from the carboxyl end of a fatty acidsubstrate. A Δ17-desaturase is also regarded as an ω3-desaturase if itacts on a C20 substrate to introduce a desaturation at the ω3 bond.

In a preferred embodiment, the Δ12-desaturase and/or Δ15-desaturase is afungal Δ12-desaturase or fungal Δ15-desaturase. As used herein, a“fungal Δ12-desaturase” or “a fungal Δ15-desaturase” refers to aΔ12-desaturase or Δ15-desaturase which is from a fungal source,including an oomycete source, or a variant thereof whose amino acidsequence is at least 95% identical thereto. Genes encoding numerousdesaturases have been isolated from fungal sources. U.S. Pat. No.7,211,656 describes a Δ12 desaturase from Saprolegnia diclina.WO2009016202 describes fungal desaturases from Helobdella robusta,Laccaria bicolor, Lottia gigantea, Microcoleus chthonoplastes, Monosigabrevicollis, Mycosphaerella fijiensis, Mycospaerella graminicola,Naegleria gruben, Nectria haematococca, Nematostella vectensis,Phycomyces blakesleeanus, Trichoderma resii, Physcomitrella patens,Postia placenta, Selaginella moellendorfii and Microdochium nivale.WO2005/012316 describes a Δ12-desaturase from Thalassiosira pseudonanaand other fungi. WO2003/099216 describes genes encoding fungalΔ12-desaturases and Δ15-desaturases isolated from Neurospora crassa,Aspergillus nidulans, Botrytis cinerea and Mortierella alpina.WO2007133425 describes fungal Δ15 desaturases isolated from:Saccharomyces kluyveri, Mortierella alpina, Aspergillus nidulans,Neurospora crassa, Fusarium graminearum, Fusarium moniliforme andMagnaporthe grisea. A preferred Δ12 desaturase is from Phytophthorasojae (Ruiz-Lopez et al., 2012).

A distinct subclass of fungal Δ12-desaturases, and of fungalΔ15-desaturases, are the bifunctional fungal Δ12/Δ15-desaturases. Genesencoding these have been cloned from Fusarium monoliforme (Accession No.DQ272516, Damude et al., 2006), Acanthamoeba castellanii (Accession No.EF017656, Sayanova et al., 2006), Perkinsus marinus (WO2007042510),Claviceps purpurea (Accession No. EF536898, Meesapyodsuk et al., 2007)and Coprinus cinereus (Accession No. AF269266, Zhang et al., 2007).

In another embodiment, the ω3-desaturase has at least some activity on,preferably greater activity on, an acyl-CoA substrate than acorresponding acyl-PC substrate. As used herein, a “correspondingacyl-PC substrate” refers to the fatty acid esterified at the sn-2position of phosphatidylcholine (PC) where the fatty acid is the samefatty acid as in the acyl-CoA substrate. For example, the acyl-CoAsubstrate may be ARA-CoA and the corresponding acyl-PC substrate is sn-2ARA-PC. In an embodiment, the activity is at least two-fold greater.Preferably, the ω3-desaturase has at least some activity on both anacyl-CoA substrate and its corresponding acyl-PC substrate and hasactivity on both C18 and C20 substrates. Examples of such ω3-desaturasesare known amongst the cloned fungal desaturases listed above.

In a further embodiment, the ω3-desaturase comprises amino acids havinga sequence as provided in SEQ ID NO:12, a biologically active fragmentthereof, or an amino acid sequence which is at least 60% identical toSEQ ID NO:12, preferably at least 90% or at least 95% identical to SEQID NO:12.

In yet a further embodiment, a desaturase for use in the presentinvention has greater activity on an acyl-CoA substrate than acorresponding acyl-PC substrate. In another embodiment, a desaturase foruse in the present invention has greater activity on an acyl-PCsubstrate than a corresponding acyl-CoA substrate, but has some activityon both substrates. As outlined above, a “corresponding acyl-PCsubstrate” refers to the fatty acid esterified at the sn-2 position ofphosphatidylcholine (PC) where the fatty acid is the same fatty acid asin the acyl-CoA substrate. In an embodiment, the greater activity is atleast two-fold greater. In an embodiment, the desaturase is a Δ5 orΔ6-desaturase, or an ω3-desaturase, examples of which are provided, butnot limited to, those listed in Table 2. To test which substrate adesaturase acts on, namely an acyl-CoA or an acyl-PC substrate, assayscan be carried out in yeast cells as described in Domergue et al. (2003)and (2005). Acyl-CoA substrate capability for a desaturase can also beinferred when an elongase, when expressed together with the desturase,has an enzymatic conversion efficiency in plant cells of at least about90% where the elongase catalyses the elongation of the product of thedesaturase. On this basis, the Δ5-desaturase and Δ4-desaturasesexpressed from the GA7 construct (Examples 2 and 3) and variants thereof(Example 5) are capable of desaturating their respective acyl-CoAsubstrates, ETA-CoA and DPA-CoA.

Elongases

Biochemical evidence suggests that the fatty acid elongation consists of4 steps: condensation, reduction, dehydration and a second reduction. Inthe context of this invention, an “elongase” refers to the polypeptidethat catalyses the condensing step in the presence of the other membersof the elongation complex, under suitable physiological conditions. Ithas been shown that heterologous or homologous expression in a cell ofonly the condensing component (“elongase”) of the elongation proteincomplex is required for the elongation of the respective acyl chain.Thus, the introduced elongase is able to successfully recruit thereduction and dehydration activities from the transgenic host to carryout successful acyl elongations. The specificity of the elongationreaction with respect to chain length and the degree of desaturation offatty acid substrates is thought to reside in the condensing component.This component is also thought to be rate limiting in the elongationreaction.

As used herein, a “Δ5-elongase” is at least capable of converting EPA toDPA. Examples of Δ5-elongases include those disclosed in WO2005/103253.In one embodiment, the Δ5-elongase has activity on EPA to produce DPAwith an efficiency of at least 60%, more preferably at least 65%, morepreferably at least 70% or most preferably at least 80% or 90%. In afurther embodiment, the Δ5-elongase comprises an amino acid sequence asprovided in SEQ ID NO:37, a biologically active fragment thereof, or anamino acid sequence which is at least 47% identical to SEQ ID NO:37. Ina further embodiment, the Δ6-elongase is from Ostreococcus taurii orOstreococcus lucimarinus (US2010/088776).

As used herein, a “Δ6-elongase” is at least capable of converting SDA toETA. Examples of Δ6-elongases include those listed in Table 1. In oneembodiment, the elongase comprises amino acids having a sequence asprovided in SEQ ID NO:25, a biologically active fragment thereof (suchas the fragment provided as SEQ ID NO:26), or an amino acid sequencewhich is at least 55% identical to one or both of SEQ ID NO:25 or SEQ IDNO:26. In an embodiment, the Δ6-elongase is from Physcomitrella patens(Zank et al., 2002; Accession No. AF428243) or Thalassiosira pseudonana(Ruiz-Lopez et al., 2012).

As used herein, a “Δ9-elongase” is at least capable of converting ALA toETrA. Examples of Δ9-elongases include those listed in Table 1. In oneembodiment, the Δ9-elongase comprises amino acids having a sequence asprovided in SEQ ID NO:43, a biologically active fragment thereof, or anamino acid sequence which is at least 80% identical to SEQ ID NO:43. Inanother embodiment, the Δ9-elongase comprises amino acids having asequence as provided in SEQ ID NO:46, a biologically active fragmentthereof, or an amino acid sequence which is at least 81% identical toSEQ ID NO:46. In another embodiment, the Δ9-elongase comprises aminoacids having a sequence as provided in SEQ ID NO:48, a biologicallyactive fragment thereof, or an amino acid sequence which is at least 50%identical to SEQ ID NO:48. In another embodiment, the Δ9-elongasecomprises amino acids having a sequence as provided in SEQ ID NO:50, abiologically active fragment thereof, or an amino acid sequence which isat least 50% identical to SEQ ID NO:50. In a further embodiment, theΔ9-elongase has greater activity on an ω6 substrate than thecorresponding ω3 substrate, or the converse.

As used herein, the term “has greater activity on an ω6 substrate thanthe corresponding ω3 substrate” refers to the relative activity of theenzyme on substrates that differ by the action of an ω3 desaturase.Preferably, the ω6 substrate is LA and the ω3 substrate is ALA.

An elongase with Δ6-elongase and Δ9-elongase activity is at leastcapable of (i) converting SDA to ETA and (ii) converting ALA to ETrA andhas greater Δ6-elongase activity than Δ9-elongase activity. In oneembodiment, the elongase has an efficiency of conversion on SDA toproduce ETA which is at least 50%, more preferably at least 60%, and/oran efficiency of conversion on ALA to produce ETrA which is at least 6%or more preferably at least 9%. In another embodiment, the elongase hasat least about 6.5 fold greater Δ6-elongase activity than Δ9-elongaseactivity. In a further embodiment, the elongase has no detectableΔ5-elongase activity

Other Enzymes

As used herein, the term “1-acyl-glycerol-3-phosphate acyltransferase”(LPAAT), also termed lysophosphatidic acid-acyltransferase oracylCoA-lysophosphatidate-acyltransferase, refers to a protein whichacylates sn-1-acyl-glycerol-3-phosphate (sn-1 G-3-P) at the sn-2position to form phosphatidic acid (PA). Thus, the term“1-acyl-glycerol-3-phosphate acyltransferase activity” refers to theacylation of (sn-1 G-3-P) at the sn-2 position to produce PA (EC2.3.1.51). Preferred LPAATs are those that can use a polyunsaturated C22acyl-CoA as substrate to transfer the polyunsaturated C22 acyl group tothe sn-2 position of LPA, forming PA. Such LPAATs are exemplified inExample 13 and can be tested as described therein. In an embodiment, anLPAAT useful for the invention comprises amino acids having a sequenceas provided in any one of SEQ ID NOs: 63 to 69, a biologically activefragment thereof, or an amino acid sequence which is at least 40%identical to any one or more of SEQ ID NOs: 63 to 69. In a preferredembodiment, an LPAAT useful for the invention comprises amino acidshaving a sequence as provided in any one of SEQ ID NOs: 64, 65 and 67, abiologically active fragment thereof, or an amino acid sequence which isat least 40% identical to any one or more of SEQ ID NOs: 64, 65 and 67.

As used herein, the term “diacylglycerol acyltransferase” (EC 2.3.1.20;DGAT) refers to a protein which transfers a fatty acyl group fromacyl-CoA to a diacylglycerol substrate to produce a triacylglycerol.Thus, the term “diacylglycerol acyltransferase activity” refers to thetransfer of acyl-CoA to diacylglycerol to produce triacylglycerol. Thereare three known types of DGAT referred to as DGAT1, DGAT2 and DGAT3respectively. DGAT1 polypeptides typically have 10 transmembranedomains, DGAT2 typically have 2 transmembrane domains, whilst DGAT3 istypically soluble. Examples of DGAT1 polypeptides include polypeptidesencoded by DGAT1 genes from Aspergillus fumigatus (Accession No.XP_755172), Arabidopsis thaliana (CAB44774), Ricinus communis(AAR11479), Vernicia fordii (ABC94472), Vernonia galamensis (ABV21945,ABV21946), Euonymus alatus (AAV31083), Caenorhabditis elegans(AAF82410), Rattus norvegicus (NP_445889), Homo sapiens (NP_036211), aswell as variants and/or mutants thereof. Examples of DGAT2 polypeptidesinclude polypeptides encoded by DGAT2 genes from Arabidopsis thaliana(Accession No. NP_566952), Ricinus communis (AAY16324), Vernicia fordii(ABC94474), 5 Mortierella ramanniana (AAK84179), Homo sapiens (Q96PD7,Q58HT₅), Bos taurus (Q70VD8), Mus musculus (AAK84175), MicromonasCCMP1545, as well as variants and/or mutants thereof. Examples of DGAT3polypeptides include polypeptides encoded by DGAT3 genes from peanut(Arachis hypogaea, Saha, et al., 2006), as well as variants and/ormutants thereof.

Polypeptides/Peptides

The term “recombinant” in the context of a polypeptide refers to thepolypeptide when produced by a cell, or in a cell-free expressionsystem, in an altered amount or at an altered rate, compared to itsnative state if it is produced naturally. In one embodiment the cell isa cell that does not naturally produce the polypeptide. However, thecell may be a cell which comprises a non-endogenous gene that causes analtered amount of the polypeptide to be produced. A recombinantpolypeptide of the invention includes polypeptides in the cell, tissue,organ or organism, or cell-free expression system, in which it isproduced i.e. a polypeptide which has not been purified or separatedfrom other components of the transgenic (recombinant) cell in which itwas produced, and polypeptides produced in such cells or cell-freesystems which are subsequently purified away from at least some othercomponents.

The terms “polypeptide” and “protein” are generally usedinterchangeably.

A polypeptide or class of polypeptides may be defined by the extent ofidentity (% identity) of its amino acid sequence to a reference aminoacid sequence, or by having a greater % identity to one reference aminoacid sequence than to another. The % identity of a polypeptide to areference amino acid sequence is typically determined by GAP analysis(Needleman and Wunsch, 1970; GCG program) with parameters of a gapcreation penalty=5, and a gap extension penalty=0.3. The query sequenceis at least 15 amino acids in length, and the GAP analysis aligns thetwo sequences over a region of at least 15 amino acids. More preferably,the query sequence is at least 50 amino acids in length, and the GAPanalysis aligns the two sequences over a region of at least 50 aminoacids. More preferably, the query sequence is at least 100 amino acidsin length and the GAP analysis aligns the two sequences over a region ofat least 100 amino acids. Even more preferably, the query sequence is atleast 250 amino acids in length and the GAP analysis aligns the twosequences over a region of at least 250 amino acids. Even morepreferably, the GAP analysis aligns two sequences over their entirelength. The polypeptide or class of polypeptides may have the sameenzymatic activity as, or a different activity than, or lack theactivity of, the reference polypeptide. Preferably, the polypeptide hasan enzymatic activity of at least 10%, at least 50%, at least 75% or atleast 90%, of the activity of the reference polypeptide.

As used herein a “biologically active” fragment is a portion of apolypeptide defined herein which maintains a defined activity of afull-length reference polypeptide, for example possessing desaturaseand/or elongase activity or other enzyme activity. Biologically activefragments as used herein exclude the full-length polypeptide.Biologically active fragments can be any size portion as long as theymaintain the defined activity. Preferably, the biologically activefragment maintains at least 10%, at least 50%, at least 75% or at least90%, of the activity of the full length protein.

With regard to a defined polypeptide or enzyme, it will be appreciatedthat % identity figures higher than those provided herein will encompasspreferred embodiments. Thus, where applicable, in light of the minimum %identity figures, it is preferred that the polypeptide/enzyme comprisesan amino acid sequence which is at least 60%, more preferably at least65%, more preferably at least 70%, more preferably at least 75%, morepreferably at least 76%, more preferably at least 80%, more preferablyat least 85%, more preferably at least 90%, more preferably at least91%, more preferably at least 92%, more preferably at least 93%, morepreferably at least 94%, more preferably at least 95%, more preferablyat least 96%, more preferably at least 97%, more preferably at least98%, more preferably at least 99%, more preferably at least 99.1%, morepreferably at least 99.2%, more preferably at least 99.3%, morepreferably at least 99.4%, more preferably at least 99.5%, morepreferably at least 99.6%, more preferably at least 99.7%, morepreferably at least 99.8%, and even more preferably at least 99.9%identical to the relevant nominated SEQ ID NO.

Amino acid sequence variants/mutants of the polypeptides of the definedherein can be prepared by introducing appropriate nucleotide changesinto a nucleic acid defined herein, or by in vitro synthesis of thedesired polypeptide. Such variants/mutants include, for example,deletions, insertions or substitutions of residues within the amino acidsequence. A combination of deletion, insertion and substitution can bemade to arrive at the final construct, provided that the final peptideproduct possesses the desired enzyme activity.

Mutant (altered) peptides can be prepared using any technique known inthe art. For example, a polynucleotide defined herein can be subjectedto in vitro mutagenesis or DNA shuffling techniques as broadly describedby Harayama (1998). Products derived from mutated/altered DNA canreadily be screened using techniques described herein to determine ifthey possess, for example, desaturase or elongase activity.

In designing amino acid sequence mutants, the location of the mutationsite and the nature of the mutation will depend on characteristic(s) tobe modified. The sites for mutation can be modified individually or inseries, e.g., by (1) substituting first with conservative amino acidchoices and then with more radical selections depending upon the resultsachieved, (2) deleting the target residue, or (3) inserting otherresidues adjacent to the located site.

Amino acid sequence deletions generally range from about 1 to 15residues, more preferably about 1 to 10 residues and typically about 1to 5 contiguous residues.

Substitution mutants have at least one amino acid residue in thepolypeptide molecule removed and a different residue inserted in itsplace. The sites of greatest interest for substitutional mutagenesisinclude sites which are not conserved amongst naturally occurringdesaturases or elongases. These sites are preferably substituted in arelatively conservative manner in order to maintain enzyme activity.Such conservative substitutions are shown in Table 3 under the headingof “exemplary substitutions”.

In a preferred embodiment a mutant/variant polypeptide has only, or notmore than, one or two or three or four conservative amino acid changeswhen compared to a naturally occurring polypeptide. Details ofconservative amino acid changes are provided in Table 3. As the skilledperson would be aware, such minor changes can reasonably be predictednot to alter the activity of the polypeptide when expressed in arecombinant cell.

Polypeptides can be produced in a variety of ways, including productionand recovery of natural polypeptides or recombinant polypeptidesaccording to methods known in the art. In one embodiment, a recombinantpolypeptide is produced by culturing a cell capable of expressing thepolypeptide under conditions effective to produce the polypeptide, suchas a host cell defined herein. A more preferred cell to produce thepolypeptide is a cell in a plant, especially in a seed in a plant.

Polynucleotides

The invention also provides and/or uses polynucleotides which may be,for example, a gene, an isolated polynucleotide, a chimeric geneticconstruct such as a T-DNA molecule, or a chimeric DNA. It may be DNA orRNA of genomic or synthetic origin, double-stranded or single-stranded,and combined with carbohydrate, lipids, protein or other materials toperform a particular activity defined herein. The term “polynucleotide”is used interchangeably herein with the term “nucleic acid molecule”. By“isolated polynucleotide” we mean a polynucleotide which, if obtainedfrom a natural source, has been separated from the polynucleotidesequences with which it is associated or linked in its native state, ora non-naturally occurring polynucleotide. Preferably, the isolatedpolynucleotide is at least 60% free, more preferably at least 75% free,and more preferably at least 90% free from other components with whichit is naturally associated.

TABLE 3 Exemplary substitutions. Original Exemplary ResidueSubstitutions Ala (A) val; leu; ile; gly Arg (R) lys Asn (N) gln; hisAsp (D) glu Cys (C) ser Gln (Q) asn; his Glu (E) asp Gly (G) pro, alaHis (H) asn; gln Ile (I) leu; val; ala Leu (L) ile; val; met; ala; pheLys (K) arg Met (M) leu; phe Phe (F) leu; val; ala Pro (P) gly Ser (S)thr Thr (T) ser Trp (W) tyr Tyr (Y) trp; phe Val (V) ile; leu; met; phe,ala

In an embodiment, a polynucleotide of the invention is non-naturallyoccurring. Examples of non-naturally occurring polynucleotides include,but are not limited to, those that have been mutated (such as by usingmethods described herein), and polynucleotides where an open readingframe encoding a protein is operably linked to a promoter to which it isnot naturally associated (such as in the constructs described herein).

As used herein, the term “gene” is to be taken in its broadest contextand includes the deoxyribonucleotide sequences comprising thetranscribed region and, if translated, the protein coding region, of astructural gene and including sequences located adjacent to the codingregion on both the 5′ and 3′ ends for a distance of at least about 2 kbon either end and which are involved in expression of the gene. In thisregard, the gene includes control signals such as promoters, enhancers,termination and/or polyadenylation signals that are naturally associatedwith a given gene, or heterologous control signals in which case thegene is referred to as a “chimeric gene”. The sequences which arelocated 5′ of the protein coding region and which are present on themRNA are referred to as 5′ non-translated sequences. The sequences whichare located 3′ or downstream of the protein coding region and which arepresent on the mRNA are referred to as 3′ non-translated sequences. Theterm “gene” encompasses both cDNA and genomic forms of a gene. A genomicform or clone of a gene contains the coding region which may beinterrupted with non-coding sequences termed “introns” or “interveningregions” or “intervening sequences.” Introns are segments of a genewhich are transcribed into nuclear RNA (hnRNA). Introns may containregulatory elements such as enhancers. Introns are removed or “splicedout” from the nuclear or primary transcript; introns therefore areabsent in the messenger RNA (mRNA) transcript. The mRNA functions duringtranslation to specify the sequence or order of amino acids in a nascentpolypeptide. The term “gene” includes a synthetic or fusion moleculeencoding all or part of the proteins of the invention described hereinand a complementary nucleotide sequence to any one of the above.

As used herein, a “chimeric DNA” or “chimeric genetic construct” refersto any DNA molecule that is not a native DNA molecule in its nativelocation, also referred to herein as a “DNA construct”. Typically, achimeric DNA or chimeric gene comprises regulatory and transcribed orprotein coding sequences that are not found operably linked together innature i.e. that are heterologous with respect to each other.Accordingly, a chimeric DNA or chimeric gene may comprise regulatorysequences and coding sequences that are derived from different sources,or regulatory sequences and coding sequences derived from the samesource, but arranged in a manner different than that found in nature.

The term “endogenous” is used herein to refer to a substance that isnormally present or produced in, for example, an unmodified plant at thesame developmental stage as the plant under investigation. An“endogenous gene” refers to a native gene in its natural location in thegenome of an organism. As used herein, “recombinant nucleic acidmolecule”, “recombinant polynucleotide” or variations thereof refer to anucleic acid molecule which has been constructed or modified byrecombinant DNA technology. The terms “foreign polynucleotide” or“exogenous polynucleotide” or “heterologous polynucleotide” and the likerefer to any nucleic acid which is introduced into the genome of a cellby experimental manipulations. Foreign or exogenous genes may be genesthat are inserted into a non-native organism, native genes introducedinto a new location within the native host, or chimeric genes. A“transgene” is a gene that has been introduced into the genome by atransformation procedure. The terms “genetically modified”, “transgenic”and variations thereof include introducing genes into cells bytransformation or transduction, mutating genes in cells and altering ormodulating the regulation of a gene in a cell or organisms to whichthese acts have been done or their progeny. A “genomic region” as usedherein refers to a position within the genome where a transgene, orgroup of transgenes (also referred to herein as a cluster), have beeninserted into a cell, or an ancestor thereof. Such regions only comprisenucleotides that have been incorporated by the intervention of man suchas by methods described herein.

The term “exogenous” in the context of a polynucleotide refers to thepolynucleotide when present in a cell in an altered amount compared toits native state. In one embodiment, the cell is a cell that does notnaturally comprise the polynucleotide. However, the cell may be a cellwhich comprises a non-endogenous polynucleotide resulting in an alteredamount of production of the encoded polypeptide. An exogenouspolynucleotide of the invention includes polynucleotides which have notbeen separated from other components of the transgenic (recombinant)cell, or cell-free expression system, in which it is present, andpolynucleotides produced in such cells or cell-free systems which aresubsequently purified away from at least some other components. Theexogenous polynucleotide (nucleic acid) can be a contiguous stretch ofnucleotides existing in nature, or comprise two or more contiguousstretches of nucleotides from different sources (naturally occurringand/or synthetic) joined to form a single polynucleotide. Typically suchchimeric polynucleotides comprise at least an open reading frameencoding a polypeptide of the invention operably linked to a promotersuitable of driving transcription of the open reading frame in a cell ofinterest.

As used herein, the term “different exogenous polynucleotides” orvariations thereof means that the nucleotide sequence of eachpolynucleotide are different by at least one, preferably more,nucleotides. The polynucleotides encode RNAs which may or may not betranslated to a protein within the cell. In an example, it is preferredthat each polynucleotide encodes a protein with a different activity. Inanother example, each exogenous polynucleotide is less than 95%, lessthan 90%, or less than 80% identical to the other exogenouspolynuclotides. Preferably, the exogenous polynucleotides encodefunctional proteins/enzymes. Furthermore, it is preferred that thedifferent exogenous polynucleotides are non-overlapping in that eachpolynucleotide is a distinct region of the, for example,extrachromosomal transfer nucleic acid which does not overlap withanother exogenous polynucleotide. At a minimum, each exogenouspolnucleotide has a transcription start and stop site, as well as thedesignated promoter. An individual exogenous polynucloeotide may or maynot comprise introns.

With regard to the defined polynucleotides, it will be appreciated that% identity figures higher than those provided above will encompasspreferred embodiments. Thus, where applicable, in light of the minimum %identity figures, it is preferred that the polynucleotide comprises apolynucleotide sequence which is at least 60%, more preferably at least65%, more preferably at least 70%, more preferably at least 75%, morepreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, more preferably at least 91%, more preferably at least92%, more preferably at least 93%, more preferably at least 94%, morepreferably at least 95%, more preferably at least 96%, more preferablyat least 97%, more preferably at least 98%, more preferably at least99%, more preferably at least 99.1%, more preferably at least 99.2%,more preferably at least 99.3%, more preferably at least 99.4%, morepreferably at least 99.5%, more preferably at least 99.6%, morepreferably at least 99.7%, more preferably at least 99.8%, and even morepreferably at least 99.9% identical to the relevant nominated SEQ ID NO.

A polynucleotide of the present invention may selectively hybridise,under stringent conditions, to a polynucleotide that encodes apolypeptide of the present invention. As used herein, stringentconditions are those that (1) employ during hybridisation a denaturingagent such as formamide, for example, 50% (v/v) formamide with 0.1%(w/v) bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodiumcitrate at 42° C.; or (2) employ 50% formamide, 5×SSC (0.75 M NaCl,0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodiumpyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50g/ml), 0.1% SDS and 10% dextran sulfate at 42° C. in 0.2×SSC and 0.1%SDS and/or (3) employ low ionic strength and high temperature forwashing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at50° C.

Polynucleotides of the invention may possess, when compared to naturallyoccurring molecules, one or more mutations which are deletions,insertions, or substitutions of nucleotide residues. Polynucleotideswhich have mutations relative to a reference sequence can be eithernaturally occurring (that is to say, isolated from a natural source) orsynthetic (for example, by performing site-directed mutagenesis or DNAshuffling on the nucleic acid as described above). It is thus apparentthat polynucleotides of the invention can be either from a naturallyoccurring source or recombinant. Preferred polynucleotides are thosewhich have coding regions that are codon-optimised for translation inplant cells, as is known in the art.

Recombinant Vectors

One embodiment of the present invention includes a recombinant vector,which comprises at least one polynucleotide molecule defined herein,inserted into any vector capable of delivering the polynucleotidemolecule into a host cell. Recombinant vectors include expressionvectors. Recombinant vectors contain heterologous polynucleotidesequences, that is, polynucleotide sequences that are not naturallyfound adjacent to polynucleotide molecules defined herein thatpreferably are derived from a species other than the species from whichthe polynucleotide molecule(s) are derived. The vector can be either RNAor DNA and typically is a plasmid. Plasmid vectors typically includeadditional nucleic acid sequences that provide for easy selection,amplification, and transformation of the expression cassette inprokaryotic cells, e.g., pUC-derived vectors, pSK-derived vectors,pGEM-derived vectors, pSP-derived vectors, pBS-derived vectors, orpreferably binary vectors containing one or more T-DNA regions.Additional nucleic acid sequences include origins of replication toprovide for autonomous replication of the vector, selectable markergenes, preferably encoding antibiotic or herbicide resistance, uniquemultiple cloning sites providing for multiple sites to insert nucleicacid sequences or genes encoded in the nucleic acid construct, andsequences that enhance transformation of prokaryotic and eukaryotic(especially plant) cells. The recombinant vector may comprise more thanone polynucleotide defined herein, for example three, four, five or sixpolynucleotides defined herein in combination, preferably a chimericgenetic construct of the invention, each polynucleotide being operablylinked to expression control sequences that are operable in the cell ofinterest. More than one polynucleotide defined herein, for example 3, 4,5 or 6 polynucleotides, are preferably covalently joined together in asingle recombinant vector, preferably within a single T-DNA molecule,which may then be introduced as a single molecule into a cell to form arecombinant cell according to the invention, and preferably integratedinto the genome of the recombinant cell, for example in a transgenicplant. Thereby, the polynucleotides which are so joined will beinherited together as a single genetic locus in progeny of therecombinant cell or plant. The recombinant vector or plant may comprisetwo or more such recombinant vectors, each containing multiplepolynucleotides, for example wherein each recombinant vector comprises3, 4, 5 or 6 polynucleotides.

“Operably linked” as used herein refers to a functional relationshipbetween two or more nucleic acid (e.g., DNA) segments. Typically, itrefers to the functional relationship of transcriptional regulatoryelement (promoter) to a transcribed sequence. For example, a promoter isoperably linked to a coding sequence, such as a polynucleotide definedherein, if it stimulates or modulates the transcription of the codingsequence in an appropriate cell. Generally, promoter transcriptionalregulatory elements that are operably linked to a transcribed sequenceare physically contiguous to the transcribed sequence, i.e., they arecis-acting. However, some transcriptional regulatory elements, such asenhancers, need not be physically contiguous or located in closeproximity to the coding sequences whose transcription they enhance.

When there are multiple promoters present, each promoter mayindependently be the same or different.

Recombinant molecules such as the chimeric DNAs or genetic constructsmay also contain (a) one or more secretory signals which encode signalpeptide sequences, to enable an expressed polypeptide defined herein tobe secreted from the cell that produces the polypeptide or which providefor localisation of the expressed polypeptide, for example for retentionof the polypeptide in the endoplasmic reticulum (ER) in the cell ortransfer into a plastid, and/or (b) contain fusion sequences which leadto the expression of nucleic acid molecules as fusion proteins. Examplesof suitable signal segments include any signal segment capable ofdirecting the secretion or localisation of a polypeptide defined herein.Recombinant molecules may also include intervening and/or untranslatedsequences surrounding and/or within the nucleic acid sequences ofnucleic acid molecules defined herein.

To facilitate identification of transformants, the nucleic acidconstruct desirably comprises a selectable or screenable marker gene as,or in addition to, the foreign or exogenous polynucleotide. By “markergene” is meant a gene that imparts a distinct phenotype to cellsexpressing the marker gene and thus allows such transformed cells to bedistinguished from cells that do not have the marker. A selectablemarker gene confers a trait for which one can “select” based onresistance to a selective agent (e.g., a herbicide, antibiotic,radiation, heat, or other treatment damaging to untransformed cells). Ascreenable marker gene (or reporter gene) confers a trait that one canidentify through observation or testing, i.e., by “screening” (e.g.,β-glucuronidase, luciferase, GFP or other enzyme activity not present inuntransformed cells). The marker gene and the nucleotide sequence ofinterest do not have to be linked. The actual choice of a marker is notcrucial as long as it is functional (i.e., selective) in combinationwith the cells of choice such as a plant cell.

Examples of bacterial selectable markers are markers that conferantibiotic resistance such as ampicillin, erythromycin, chloramphenicolor tetracycline resistance, preferably kanamycin resistance. Exemplaryselectable markers for selection of plant transformants include, but arenot limited to, a hyg gene which encodes hygromycin B resistance; aneomycin phosphotransferase (nptII) gene conferring resistance tokanamycin, paromomycin, G418; a glutathione-S-transferase gene from ratliver conferring resistance to glutathione derived herbicides as, forexample, described in EP 256223; a glutamine synthetase gene conferring,upon overexpression, resistance to glutamine synthetase inhibitors suchas phosphinothricin as, for example, described in WO 87/05327, anacetyltransferase gene from Streptomyces viridochromogenes conferringresistance to the selective agent phosphinothricin as, for example,described in EP 275957, a gene encoding a 5-enolshikimate-3-phosphatesynthase (EPSPS) conferring tolerance to N-phosphonomethylglycine as,for example, described by Hinchee et al. (1988), a bar gene conferringresistance against bialaphos as, for example, described in WO91/02071; anitrilase gene such as bxn from Klebsiella ozaenae which confersresistance to bromoxynil (Stalker et al., 1988); a dihydrofolatereductase (DHFR) gene conferring resistance to methotrexate (Thillet etal., 1988); a mutant acetolactate synthase gene (ALS), which confersresistance to imidazolinone, sulfonylurea or other ALS-inhibitingchemicals (EP 154,204); a mutated anthranilate synthase gene thatconfers resistance to 5-methyl tryptophan; or a dalapon dehalogenasegene that confers resistance to the herbicide.

Preferred screenable markers include, but are not limited to, a uidAgene encoding a β-glucuronidase (GUS) enzyme for which variouschromogenic substrates are known, a green fluorescent protein gene(Niedz et al., 1995) or derivatives thereof, a luciferase (luc) gene (Owet al., 1986), which allows for bioluminescence detection, and othersknown in the art. By “reporter molecule” as used in the presentspecification is meant a molecule that, by its chemical nature, providesan analytically identifiable signal that facilitates determination ofpromoter activity by reference to protein product.

Preferably, the nucleic acid construct is stably incorporated into thegenome of 5 the cell, such as the plant cell. Accordingly, the nucleicacid may comprise appropriate elements which allow the molecule to beincorporated into the genome, preferably the right and left bordersequences of a T-DNA molecule, or the construct is placed in anappropriate vector which can be incorporated into a chromosome of thecell.

Expression

As used herein, an expression vector is a DNA vector that is capable oftransforming a host cell and of effecting expression of one or morespecified polynucleotide molecule(s). Preferred expression vectors ofthe present invention can direct gene expression in yeast and/or plantcells. Expression vectors useful for the invention contain regulatorysequences such as transcription control sequences, translation controlsequences, origins of replication, and other regulatory sequences thatare compatible with the recombinant cell and that control the expressionof polynucleotide molecules of the present invention. In particular,polynucleotides or vectors useful for the present invention includetranscription control sequences. Transcription control sequences aresequences which control the initiation, elongation, and termination oftranscription. Particularly important transcription control sequencesare those which control transcription initiation, such as promoter andenhancer sequences. Suitable transcription control sequences include anytranscription control sequence that can function in at least one of therecombinant cells of the present invention. The choice of the regulatorysequences used depends on the target organism such as a plant and/ortarget organ or tissue of interest. Such regulatory sequences may beobtained from any eukaryotic organism such as plants or plant viruses,or may be chemically synthesized. A variety of such transcriptioncontrol sequences are known to those skilled in the art. Particularlypreferred transcription control sequences are promoters active indirecting transcription in plants, either constitutively or stage and/ortissue specific, depending on the use of the plant or parts thereof.

A number of vectors suitable for stable transfection of plant cells orfor the establishment of transgenic plants have been described in, e.g.,Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987;Weissbach and Weissbach, Methods for Plant Molecular Biology, AcademicPress, 1989; and Gelvin et al., Plant Molecular Biology Manual, KluwerAcademic Publishers, 1990. Typically, plant expression vectors include,for example, one or more cloned plant genes under the transcriptionalcontrol of 5′ and 3′ regulatory sequences and a dominant selectablemarker. Such plant expression vectors also can contain a promoterregulatory region (e.g., a regulatory region controlling inducible orconstitutive, environmentally- or developmentally-regulated, or cell- ortissue-specific expression), a transcription initiation start site, aribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal.

A number of constitutive promoters that are active in plant cells havebeen described. Suitable promoters for constitutive expression in plantsinclude, but are not limited to, the cauliflower mosaic virus (CaMV) 35Spromoter, the Figwort mosaic virus (FMV) 35S, the sugarcane bacilliformvirus promoter, the commelina yellow mottle virus promoter, thelight-inducible promoter from the small subunit of theribulose-1,5-bis-phosphate carboxylase, the rice cytosolictriosephosphate isomerase promoter, the adeninephosphoribosyltransferase promoter of Arabidopsis, the rice actin 1 genepromoter, the mannopine synthase and octopine synthase promoters, theAdh promoter, the sucrose synthase promoter, the R gene complexpromoter, and the chlorophyll α/β binding protein gene promoter

For the purpose of expression in source tissues of the plant, such asthe leaf, seed, root or stem, it is preferred that the promotersutilized in the present invention have relatively high expression inthese specific tissues. For this purpose, one may choose from a numberof promoters for genes with tissue- or cell-specific or -enhancedexpression. Examples of such promoters reported in the literatureinclude the chloroplast glutamine synthetase GS2 promoter from pea, thechloroplast fructose-1,6-biphosphatase promoter from wheat, the nuclearphotosynthetic ST-LS 1 promoter from potato, the serine/threonine kinasepromoter and the glucoamylase (CHS) promoter from Arabidopsis thaliana.Also reported to be active in photosynthetically active tissues areribulose-1,5-bisphosphate carboxylase promoters, and Cab promoters.

A variety of plant gene promoters that are regulated in response toenvironmental, hormonal, chemical, and/or developmental signals, alsocan be used for expression of genes in plant cells, including promotersregulated by (1) heat, (2) light (e.g., pea RbcS-3A promoter, maize RbcSpromoter); (3) hormones, such as abscisic acid, (4) wounding (e.g.,WunI); or (5) chemicals, such as methyl jasmonate, salicylic acid,steroid hormones, alcohol, Safeners (WO97/06269), or it may also beadvantageous to employ (6) organ-specific promoters.

As used herein, the term “plant seed specific promoter” or variationsthereof refer to a promoter that preferentially, when compared to otherplant tissues, directs gene transcription in a developing seed of aplant. In an embodiment, the seed specific promoter is expressed atleast 5-fold more strongly in the developing seed of the plant relativeto the leaves and/or stems of the plant, and is preferably expressedmore strongly in the embryo of the developing seed compared to otherplant tissues. Preferably, the promoter only directs expression of agene of interest in the developing seed, and/or expression of the geneof interest in other parts of the plant such as leaves is not detectableby Northern blot analysis and/or RT-PCR. Typically, the promoter drivesexpression of genes during growth and development of the seed, inparticular during the phase of synthesis and accumulation of storagecompounds in the seed. Such promoters may drive gene expression in theentire plant storage organ or only part thereof such as the seedcoat, orcotyledon(s), preferably in the embryos, in seeds of dicotyledonousplants or the endosperm or aleurone layer of a seeds of monocotyledonousplants.

Preferred promoters for seed-specific expression include i) promotersfrom genes encoding enzymes involved in fatty acid biosynthesis andaccumulation in seeds, such as desaturases and elongases, ii) promotersfrom genes encoding seed storage proteins, and iii) promoters from genesencoding enzymes involved in carbohydrate biosynthesis and accumulationin seeds. Seed specific promoters which are suitable are the oilseedrape napin gene promoter (U.S. Pat. No. 5,608,152), the Vicia faba USPpromoter (Baumlein et al., 1991), the Arabidopsis oleosin promoter(WO98/45461), the Phaseolus vulgaris phaseolin promoter (U.S. Pat. No.5,504,200), the Brassica Bce4 promoter (WO91/13980) or the legumin LeB4promoter from Vicia faba (Baumlein et al., 1992), and promoters whichlead to the seed-specific expression in monocots such as maize, barley,wheat, rye, rice and the like. Notable promoters which are suitable arethe barley lpt2 or lpt1 gene promoter (WO95/15389 and WO95/23230) or thepromoters described in WO99/16890 (promoters from the barley hordeingene, the rice glutelin gene, the rice oryzin gene, the rice prolamingene, the wheat gliadin gene, the wheat glutelin gene, the maize zeingene, the oat glutelin gene, the sorghum kasirin gene, the rye secalingene). Other promoters include those described by Broun et al. (1998),Potenza et al. (2004), US20070192902 and US20030159173. In anembodiment, the seed specific promoter is preferentially expressed indefined parts of the seed such as the embryo, cotyledon(s) or theendosperm. Examples of such specific promoters include, but are notlimited to, the FP1 promoter (Ellerstrom et al., 1996), the pea leguminpromoter (Perrin et al., 2000), the bean phytohemagglutnin promoter(Perrin et al., 2000), the conlinin 1 and conlinin 2 promoters for thegenes encoding the flax 2S storage proteins (Cheng et al., 2010), thepromoter of the FAE1 gene from Arabidopsis thaliana, the BnGLP promoterof the globulin-like protein gene of Brassica napus, the LPXR promoterof the peroxiredoxin gene from Linum usitatissimum.

The 5′ non-translated leader sequence can be derived from the promoterselected to express the heterologous gene sequence of the polynucleotideof the present invention, or preferably is heterologous with respect tothe coding region of the enzyme to be produced, and can be specificallymodified if desired so as to increase translation of mRNA. For a reviewof optimizing expression of transgenes, see Koziel et al. (1996). The 5′non-translated regions can also be obtained from plant viral RNAs(Tobacco mosaic virus, Tobacco etch virus, Maize dwarf mosaic virus,Alfalfa mosaic virus, among others) from suitable eukaryotic genes,plant genes (wheat and maize chlorophyll a/b binding protein geneleader), or from a synthetic gene sequence. The present invention is notlimited to constructs wherein the non-translated region is derived fromthe 5′ non-translated sequence that accompanies the promoter sequence.The leader sequence could also be derived from an unrelated promoter orcoding sequence. Leader sequences useful in context of the presentinvention comprise the maize Hsp70 leader (U.S. Pat. No. 5,362,865 andU.S. Pat. No. 5,859,347), and the TMV omega element.

The termination of transcription is accomplished by a 3′ non-translatedDNA sequence operably linked in the chimeric vector to thepolynucleotide of interest. The 3′ non-translated region of arecombinant DNA molecule contains a polyadenylation signal thatfunctions in plants to cause the addition of adenylate nucleotides tothe 3′ end of the RNA. The 3′ non-translated region can be obtained fromvarious genes that are expressed in plant cells. The nopaline synthase3′ untranslated region, the 3′ untranslated region from pea smallsubunit Rubisco gene, the 3′ untranslated region from soybean 7S seedstorage protein gene or a flax conlinin gene are commonly used in thiscapacity. The 3′ transcribed, non-translated regions containing thepolyadenylate signal of Agrobacterium tumor-inducing (Ti) plasmid genesare also suitable.

Recombinant DNA technologies can be used to improve expression of atransformed polynucleotide molecule by manipulating, for example, thenumber of copies of the polynucleotide molecule within a host cell, theefficiency with which those polynucleotide molecules are transcribed,the efficiency with which the resultant transcripts are translated, andthe efficiency of post-translational modifications. Recombinanttechniques useful for increasing the expression of polynucleotidemolecules defined herein include, but are not limited to, integration ofthe polynucleotide molecule into one or more host cell chromosomes,addition of stability sequences to mRNAs, substitutions or modificationsof transcription control signals (e.g., promoters, operators,enhancers), substitutions or modifications of translational controlsignals (e.g., ribosome binding sites, Shine-Dalgamo sequences),modification of polynucleotide molecules to correspond to the codonusage of the host cell, and the deletion of sequences that destabilizetranscripts.

Recombinant Cells

The invention also provides a recombinant cell, preferably a recombinantplant cell, which is a host cell transformed with one or morerecombinant molecules, such as the polynucleotides, chimeric geneticconstructs or recombinant vectors defined herein. The recombinant cellmay comprise any combination thereof, such as two or three recombinantvectors, or a recombinant vector and one or more additionalpolynucleotides or chimeric DNAs. Suitable cells of the inventioninclude any cell that can be transformed with a polynucleotide, chimericDNA or recombinant vector of the invention, such as for example, amolecule encoding a polypeptide or enzyme described herein. The cell ispreferably a cell which is thereby capable of being used for producingLC-PUFA. The recombinant cell may be a cell in culture, a cell in vitro,or in an organism such as for example a plant, or in an organ such asfor example a seed or a leaf. Preferably, the cell is in a plant orplant part, more preferably in the seed of a plant.

Host cells into which the polynucleotide(s) are introduced can be eitheruntransformed cells or cells that are already transformed with at leastone nucleic acid molecule. Such nucleic acid molecules may be related toLC-PUFA synthesis, or unrelated. Host cells of the present inventioneither can be endogenously (i.e., naturally) capable of producingproteins defined herein, in which case the recombinant cell derivedtherefrom has an enhanced capability of producing the polypeptides, orcan be capable of producing such proteins only after being transformedwith at least one polynucleotide of the invention. In an embodiment, arecombinant cell of the invention has a enhanced capacity to synthesizea long chain polyunsaturated fatty acid. As used herein, the term “cellwith an enhanced capacity to synthesize a long chain polyunsaturatedfatty acid” is a relative term where the recombinant cell of theinvention is compared to the host cell lacking the polynucleotide(s) ofthe invention, with the recombinant cell producing more long chainpolyunsaturated fatty acids, or a greater concentration of LC-PUFA suchas DHA (relative to other fatty acids), than the native cell. The cellwith an enhanced capacity to synthesize another product, such as forexample another fatty acid, a lipid, a carbohydrate such as starch, anRNA molecule, a polypeptide, a pharmaceutical or other product has acorresponding meaning.

Host cells of the present invention can be any cell capable of producingat least one protein described herein, and include bacterial, fungal(including yeast), parasite, arthropod, animal and plant cells. Thecells may be prokaryotic or eukaryotic. Preferred host cells are yeastand plant cells. In a preferred embodiment, the plant cell is a seedcell, in particular a cell in a cotyledon or endosperm of a seed. In oneembodiment, the cell is an animal cell or an algal cell. The animal cellmay be of any type of animal such as, for example, a non-human animalcell, a non-human vertebrate cell, a non-human mammalian cell, or cellsof aquatic animals such as, fish or crustacea, invertebrates, insects,etc. The cells may be of an organism suitable for a fermentationprocess. As used herein, the term the “fermentation process” refers toany fermentation process or any process comprising a fermentation step.Examples of fermenting microorganisms include fungal organisms, such asyeast. As used herein, “yeast” includes Saccharomyces spp.,Saccharomyces cerevisiae, Saccharomyces carlbergensis, Candida spp.,Kluveromyces spp., Pichia spp., Hansenula spp., Trichoderma spp.,Lipomyces starkey, and Yarrowia lipolytica. Preferred yeast includestrains of the Saccharomyces spp., and in particular, Saccharomycescerevisiae.

Transgenic Plants

The invention also provides a plant comprising a cell of the invention,such as a transgenic plant comprising one or more polynucleotides of theinvention. The term “plant” as used herein as a noun refers to wholeplants, but as used as an adjective refers to any substance which ispresent in, obtained from, derived from, or related to a plant, such asfor example, plant organs (e.g. leaves, stems, roots, flowers), singlecells (e.g. pollen), seeds, plant cells and the like. The term “plantpart” refers to all plant parts that comprise the plant DNA, includingvegetative structures such as, for example, leaves or stems, roots,floral organs or structures, pollen, seed, seed parts such as an embryo,endosperm, scutellum or seed coat, plant tissue such as, for example,vascular tissue, cells and progeny of the same, as long as the plantpart synthesizes lipid according to the invention.

A “transgenic plant”, “genetically modified plant” or variations thereofrefers to a plant that contains a gene construct (“transgene”) not foundin a wild-type plant of the same species, variety or cultivar.Transgenic plants as defined in the context of the present inventioninclude plants and their progeny which have been genetically modifiedusing recombinant techniques to cause production of the lipid or atleast one polypeptide defined herein in the desired plant or plantorgan. Transgenic plant cells and transgenic plant parts havecorresponding meanings. A “transgene” as referred to herein has thenormal meaning in the art of biotechnology and includes a geneticsequence which has been produced or altered by recombinant DNA or RNAtechnology and which has been introduced into a cell of the invention,preferably a plant cell. The transgene may include genetic sequencesderived from a plant cell which may be of the same species, variety orcultivar as the plant cell into which the transgene is introduced or ofa different species, variety or cultivar, or from a cell other than aplant cell. Typically, the transgene has been introduced into the cell,such as a plant, by human manipulation such as, for example, bytransformation but any method can be used as one of skill in the artrecognizes.

The terms “seed” and “grain” are used interchangeably herein. “Grain”refers to mature grain such as harvested grain or grain which is stillon a plant but ready for harvesting, but can also refer to grain afterimbibition or germination, according to the context. Mature grain orseed commonly has a moisture content of less than about 18-20%.“Developing seed” as used herein refers to a seed prior to maturity,typically found in the reproductive structures of the plant afterfertilisation or anthesis, but can also refer to such seeds prior tomaturity which are isolated from a plant.

As used herein, the term “obtaining a plant part” or “obtaining a seed”refers to any means of obtaining a plant part or seed, respectively,including harvesting of the plant parts or seed from plants in the fieldor in containment such as a greenhouse or growth chamber, or by purchaseor receipt from a supplier of the plant parts or seed. The seed may besuitable for planting i.e. able to germinate and produce progeny plants,or alternatively has been processed in such a way that it is no longerable to germinate, e.g. cracked, polished or milled seed which is usefulfor food or feed applications, or for extraction of lipid of theinvention.

As used herein, the term “plant storage organ” refers to a part of aplant specialized to storage energy in the form of, for example,proteins, carbohydrates, fatty acids and/or oils. Examples of plantstorage organs are seed, fruit, tuberous roots, and tubers. A preferredplant storage organ of the invention is seed.

As used herein, the term “phenotypically normal” refers to a geneticallymodified plant or plant organ, particularly a storage organ such as aseed, tuber or fruit of the invention not having a significantly reducedability to grow and reproduce when compared to an unmodified plant orplant organ. In an embodiment, the genetically modified plant or plantorgan which is phenotypically normal comprises an exogenouspolynucleotide encoding a silencing suppressor operably linked to aplant storage organ specific promoter and has an ability to grow orreproduce which is essentially the same as an isogenic plant or organnot comprising said polynucleotide. Preferably, the biomass, growthrate, germination rate, storage organ size, seed size and/or the numberof viable seeds produced is not less than 90% of that of a plant lackingsaid exogenous polynucleotide when grown under identical conditions.This term does not encompass features of the plant which may bedifferent to the wild-type plant but which do not effect the usefulnessof the plant for commercial purposes such as, for example, a ballerinaphenotype of seedling leaves.

Plants provided by or contemplated for use in the practice of thepresent invention include both monocotyledons and dicotyledons. Inpreferred embodiments, the plants of the present invention are cropplants (for example, cereals and pulses, maize, wheat, potatoes,tapioca, rice, sorghum, millet, cassava, barley, or pea), or otherlegumes. The plants may be grown for production of edible roots, tubers,leaves, stems, flowers or fruit. The plants may be vegetables orornamental plants. The plants of the invention may be: corn (Zea mays),canola (Brassica napus, Brassica rapa ssp.), mustard (Brassica juncea),flax (Linum usitatissimum), alfalfa (Medicago sativa), rice (Oryzasativa), rye (Secale cerale), sorghum (Sorghum bicolour, Sorghumvulgare), sunflower (Helianthus annus), wheat (Tritium aestivum),soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanumtuberosum), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum),sweet potato (Lopmoea batatus), cassava (Manihot esculenta), coffee(Cofea spp.), coconut (Cocos nucifera), pineapple (Anana comosus),citris tree (Citrus spp.), cocoa (Theobroma cacao), tea (Camelliasenensis), banana (Musa spp.), avocado (Persea americana), fig (Ficuscasica), guava (Psidium guajava), mango (Mangifer indica), olive (Oleaeuropaea), papaya (Carica papaya), cashew (Anacardium occidentale),macadamia (Macadamia intergrifolia), almond (Prunus amygdalus), sugarbeets (Beta vulgaris), oats, or barley.

In a preferred embodiment, the plant is an angiosperm.

In an embodiment, the plant is an oilseed plant, preferably an oilseedcrop plant. As used herein, an “oilseed plant” is a plant species usedfor the commercial production of oils from the seeds of the plant. Theoilseed plant may be oil-seed rape (such as canola), maize, sunflower,soybean, sorghum, flax (linseed) or sugar beet. Furthermore, the oilseedplant may be other Brassicas, cotton, peanut, poppy, mustard, castorbean, sesame, safflower, or nut producing plants. The plant may producehigh levels of oil in its fruit, such as olive, oil palm or coconut.Horticultural plants to which the present invention may be applied arelettuce, endive, or vegetable brassicas including cabbage, broccoli, orcauliflower. The present invention may be applied in tobacco, cucurbits,carrot, strawberry, tomato, or pepper.

In a further preferred embodiment, the non-transgenic plant used toproduce a transgenic plant of the invention produces oil, especially inthe seed, which has i) less than 20%, less than 10% or less than 5% 18:2fatty acids and/or ii) less than 10% or less than 5% 18:3 fatty acids.

In a preferred embodiment, the transgenic plant is homozygous for eachand every gene that has been introduced (transgene) so that its progenydo not segregate for the desired phenotype. The transgenic plant mayalso be heterozygous for the introduced transgene(s), preferablyuniformly heterozygous for the transgene, such as for example in F1progeny which have been grown from hybrid seed. Such plants may provideadvantages such as hybrid vigor, well known in the art.

Where relevant, the transgenic plants may also comprise additionaltransgenes encoding enzymes involved in the production of LC-PUFAs suchas, but not limited to, a Δ6-desaturase, a Δ9-elongase, a Δ8-desaturase,a Δ6-elongase, a Δ5-desaturase, an ω3-desaturase, a Δ4-desaturase, aΔ5-elongase, diacylglycerol acyltransferase, LPAAT, a Δ17-desaturase, aΔ15-desaturase and/or a Δ12 desaturase. Examples of such enzymes withone of more of these activities are known in the art and include thosedescribed herein. In specific examples, the transgenic plant at leastcomprises exogenous polynucleotides encoding;

a) a Δ4-desaturase, a Δ5-desaturase, a Δ6-desaturase, a Δ5-elongase anda Δ6-elongase,

b) a Δ4-desaturase, a Δ5-desaturase, a Δ8-desaturase, a Δ5-elongase anda Δ9-elongase,

c) a Δ4-desaturase, a Δ5-desaturase, a Δ6-desaturase, a Δ5-elongase, aΔ6-elongase, and a Δ15-desaturase,

d) a Δ4-desaturase, a Δ5-desaturase, a Δ8-desaturase, a Δ5-elongase, aΔ9-elongase, and a Δ15-desaturase,

e) a Δ4-desaturase, a Δ5-desaturase, a Δ6-desaturase, a Δ5-elongase, aΔ6-elongase, and a Δ17-desaturase, or

f) a Δ4-desaturase, a Δ5-desaturase, a Δ8-desaturase, a Δ5-elongase, aΔ9-elongase, and a Δ17-desaturase.

In an embodiment, the exogenous polynucleotides encode set ofpolypeptides which are a Pythium irregulare Δ6-desaturase, aThraustochytrid Δ5-desaturase or an Emiliana huxleyi Δ5-desaturase, aPhyscomitrella patens Δ6-elongase, a Thraustochytrid Δ5-elongase or anOstreococcus taurii Δ5-elongase, a Phytophthora infestans ω3-desaturaseor a Pythium irregulare ω3-desaturase, and a ThraustochytridΔ4-desaturase.

In an embodiment, plants of the invention are grown in the field,preferably as a population of at least 1,000 or 1,000,000 plants thatare essentially the same, or in an area of at least 1 hectare. Plantingdensities differ according to the plant species, plant variety, climate,soil conditions, fertiliser rates and other factors as known in the art.For example, canola is typically grown at a planting density of 1.2-1.5million plants per hectare. Plants are harvested as is known in the art,which may comprise swathing, windrowing and/or reaping of plants,followed by threshing and/or winnowing of the plant material to separatethe seed from the remainder of the plant parts often in the form ofchaff. Alternatively, seed may be harvested from plants in the field ina single process, namely combining.

Transformation of Plants

Transgenic plants can be produced using techniques known in the art,such as those generally described in A. Slater et al., PlantBiotechnology—The Genetic Manipulation of Plants, Oxford UniversityPress (2003), and P. Christou and H. Klee, Handbook of PlantBiotechnology, John Wiley and Sons (2004).

As used herein, the terms “stably transforming”, “stably transformed”and variations thereof refer to the integration of the exogenous nucleicacid molecules into the genome of the cell such that they aretransferred to progeny cells during cell division without the need forpositively selecting for their presence. Stable transformants, orprogeny thereof, can be selected by any means known in the art such asSouthern blots on chromosomal DNA or in situ hybridization of genomicDNA.

Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because DNA can be introduced intocells in whole plant tissues or plant organs or explants in tissueculture, for either transient expression or for stable integration ofthe DNA in the plant cell genome. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art (see, for example, U.S. Pat. No. 5,177,010, U.S. Pat.No. 5,104,310, U.S. Pat. No. 5,004,863 or U.S. Pat. No. 5,159,135)including floral dipping methods using Agrobacterium or other bacteriathat can transfer DNA into plant cells. The region of DNA to betransferred is defined by the border sequences, and the intervening DNA(T-DNA) is usually inserted into the plant genome. Further, theintegration of the T-DNA is a relatively precise process resulting infew rearrangements. In those plant varieties whereAgrobacterium-mediated transformation is efficient, it is the method ofchoice because of the facile and defined nature of the gene transfer.Preferred Agrobacterium transformation vectors are capable ofreplication in E. coli as well as Agrobacterium, allowing for convenientmanipulations as described (Klee et al., In: Plant DNA InfectiousAgents, Hohn and Schell, eds., Springer-Verlag, New York, pp. 179-203(1985).

Acceleration methods that may be used include, for example,microprojectile bombardment and the like. One example of a method fordelivering transforming nucleic acid molecules to plant cells ismicroprojectile bombardment. This method has been reviewed by Yang etal., Particle Bombardment Technology for Gene Transfer, Oxford Press,Oxford, England (1994). Non-biological particles (microprojectiles) thatmay be coated with nucleic acids and delivered into cells by apropelling force. Exemplary particles include those comprised oftungsten, gold, platinum, and the like. A particular advantage ofmicroprojectile bombardment, in addition to it being an effective meansof reproducibly transforming monocots, is that neither the isolation ofprotoplasts, nor the susceptibility of Agrobacterium infection arerequired.

In another alternative embodiment, plastids can be stably transformed.Methods disclosed for plastid transformation in higher plants includeparticle gun delivery of DNA containing a selectable marker andtargeting of the DNA to the plastid genome through homologousrecombination (U.S. Pat. No. 5,451,513, U.S. Pat. No. 5,545,818, U.S.Pat. No. 5,877,402, U.S. Pat. No. 5,932,479, and WO99/05265).

Other methods of cell transformation can also be used and include butare not limited to introduction of DNA into plants by direct DNAtransfer into pollen, by direct injection of DNA into reproductiveorgans of a plant, or by direct injection of DNA into the cells ofimmature embryos followed by the rehydration of desiccated embryos.

The regeneration, development, and cultivation of plants from singleplant protoplast transformants or from various transformed explants iswell known in the art (Weissbach et al., In: Methods for Plant MolecularBiology, Academic Press, San Diego, Calif., (1988). This regenerationand growth process typically includes the steps of selection oftransformed cells, culturing those individualized cells through theusual stages of embryonic development through the rooted plantlet stage.Transgenic embryos and seeds are similarly regenerated. The resultingtransgenic rooted shoots are thereafter planted in an appropriate plantgrowth medium such as soil.

The development or regeneration of plants containing the foreign,exogenous gene is well known in the art. Preferably, the regeneratedplants are self-pollinated to provide homozygous transgenic plants.Otherwise, pollen obtained from the regenerated plants is crossed toseed-grown plants of agronomically important lines.

Conversely, pollen from plants of these important lines is used topollinate regenerated plants. A transgenic plant of the presentinvention containing a desired exogenous nucleic acid is cultivatedusing methods well known to one skilled in the art.

To confirm the presence of the transgenes in transgenic cells andplants, a polymerase chain reaction (PCR) amplification or Southern blotanalysis can be performed using methods known to those skilled in theart. Expression products of the transgenes can be detected in any of avariety of ways, depending upon the nature of the product, and includeWestern blot and enzyme assay. Once transgenic plants have beenobtained, they may be grown to produce plant tissues or parts having thedesired phenotype. The plant tissue or plant parts, may be harvested,and/or the seed collected. The seed may serve as a source for growingadditional plants with tissues or parts having the desiredcharacteristics.

A transgenic plant formed using Agrobacterium or other transformationmethods typically contains a single genetic locus on one chromosome.Such transgenic plants can be referred to as being hemizygous for theadded gene(s). More preferred is a transgenic plant that is homozygousfor the added gene(s); i.e., a transgenic plant that contains two addedgenes, one gene at the same locus on each chromosome of a chromosomepair. A homozygous transgenic plant can be obtained by self-fertilisinga hemizygous transgenic plant, germinating some of the seed produced andanalyzing the resulting plants for the gene of interest.

It is also to be understood that two different transgenic plants thatcontain two independently segregating exogenous genes or loci can alsobe crossed (mated) to produce offspring that contain both sets of genesor loci. Selfing of appropriate F1 progeny can produce plants that arehomozygous for both exogenous genes or loci. Back-crossing to a parentalplant and out-crossing with a non-transgenic plant are alsocontemplated, as is vegetative propagation. Descriptions of otherbreeding methods that are commonly used for different traits and cropscan be found in Fehr, In: Breeding Methods for Cultivar Development,Wilcox J. ed., American Society of Agronomy, Madison Wis. (1987).

Enhancing Exogenous RNA Levels and Stabilized Expression SilencingSuppressors

In an embodiment, a cell, plant or plant part of the invention comprisesan exogenous polynucleotide encoding a silencing suppressor protein.

Post-transcriptional gene silencing (PTGS) is a nucleotidesequence-specific defense mechanism that can target both cellular andviral mRNAs for degradation PTGS occurs in plants or fungi stably ortransiently transformed with foreign (heterologous) or endogenous DNAand results in the reduced accumulation of RNA molecules with sequencesimilarity to the introduced nucleic acid.

It has widely been considered that co-expression of a silencingsuppressor with a transgene of interest will increase the levels of RNApresent in the cell transcribed from the transgene. Whilst this hasproven true for cells in vitro, significant side-effects have beenobserved in many whole plant co-expression studies. More specifically,as described in Mallory et al. (2002), Chapman et al. (2004), Chen etal. (2004), Dunoyer et al. (2004), Zhang et al. (2006), Lewsey et al.(2007) and Meng et al. (2008) plants expressing silencing suppressors,generally under constitutive promoters, are often phenotypicallyabnormal to the extent that they are not useful for commercialproduction.

Recently, it has been found that RNA molecule levels can be increased,and/or RNA molecule levels stabilized over numerous generations, bylimiting the expression of the silencing suppressor to a seed of a plantor part thereof (WO2010/057246). As used herein, a “silencing suppressorprotein” or SSP is any polypeptide that can be expressed in a plant cellthat enhances the level of expression product from a different transgenein the plant cell, particularly over repeated generations from theinitially transformed plant. In an embodiment, the SSP is a viralsilencing suppressor or mutant thereof. A large number of viralsilencing suppressors are known in the art and include, but are notlimited to P19, V2, P38, Pe-Po and RPV-P0. In an embodiment, the viralsilencing suppressor comprises amino acids having a sequence as providedin any one of SEQ ID NOs 53 to 57, a biologically active fragmentthereof, or an amino acid sequence which is at least 50% identical toany one or more of SEQ ID NOs 53 to 57 and which has activity as asilencing suppressor.

As used herein, the terms “stabilising expression”, “stably expressed”,“stabilised expression” and variations thereof refer to level of the RNAmolecule being essentially the same or higher in progeny plants overrepeated generations, for example at least three, at least five or atleast 10 generations, when compared to isogenic plants lacking theexogenous polynucleotide encoding the silencing suppressor. However,this term(s) does not exclude the possibility that over repeatedgenerations there is some loss of levels of the RNA molecule whencompared to a previous generation, for example not less than a 10% lossper generation.

The suppressor can be selected from any source e.g. plant, viral, mammaletc. See WO2010/057246 for a list of viruses from which the suppressorcan be obtained and the protein (eg B2, P14 etc) or coding regiondesignation for the suppressor from each particular virus. Multiplecopies of a suppressor may be used. Different suppressors may be usedtogether (e. g., in tandem).

RNA Molecules

Essentially any RNA molecule which is desirable to be expressed in aplant seed can be co-expressed with the silencing suppressor. Theencoded polypeptides may be involved in metabolism of oil, starch,carbohydrates, nutrients, etc., or may be responsible for the synthesisof proteins, peptides, fatty acids, lipids, waxes, oils, starches,sugars, carbohydrates, flavors, odors, toxins, carotenoids. hormones,polymers, flavonoids, storage proteins, phenolic acids, alkaloids,lignins, tannins, celluloses, glycoproteins, glycolipids, etc,preferably the biosynthesis or assembly of TAG.

In a particular example, the plants produced increased levels of enzymesfor oil production in plants such as Brassicas, for example canola orsunflower, safflower, flax, cotton, soya bean, Camelina or maize.

Levels of LC-PUFA Produced

The levels of the LC-PUFA or combination of LC-PUFAs that are producedin the recombinant cell or plant part such as seed are of importance.The levels may be expressed as a composition (in percent) of the totalfatty acid that is a particular LC-PUFA or group of related LC-PUFA, forexample the ω3 LC-PUFA or the ω6 LC-PUFA, or the VLC-PUFA, or otherwhich may be determined by methods known in the art. The level may alsobe expressed as a LC-PUFA content, such as for example the percentage ofLC-PUFA in the dry weight of material comprising the recombinant cells,for example the percentage of the weight of seed that is LC-PUFA. Itwill be appreciated that the LC-PUFA that is produced in an oilseed maybe considerably higher in terms of LC-PUFA content than in a vegetableor a grain that is not grown for oil production, yet both may havesimilar LC-PUFA compositions, and both may be used as sources of LC-PUFAfor human or animal consumption.

The levels of LC-PUFA may be determined by any of the methods known inthe art. In a preferred method, total lipid is extracted from the cells,tissues or organisms and the fatty acid converted to methyl estersbefore analysis by gas chromatography (GC). Such techniques aredescribed in Example 1. The peak position in the chromatogram may beused to identify each particular fatty acid, and the area under eachpeak integrated to determine the amount. As used herein, unless statedto the contrary, the percentage of particular fatty acid in a sample isdetermined as the area under the peak for that fatty acid as apercentage of the total area for fatty acids in the chromatogram. Thiscorresponds essentially to a weight percentage (w/w). The identity offatty acids may be confirmed by GC-MS. Total lipid may be separated bytechniques known in the art to purify fractions such as the TAGfraction. For example, thin-layer chromatography (TLC) may be performedat an analytical scale to separate TAG from other lipid fractions suchas DAG, acyl-CoAs or phospholipid in order to determine the fatty acidcomposition specifically of TAG.

In one embodiment, the sum total of ARA, EPA, DPA and DHA in the fattyacids in the extracted lipid is between about 7% and about 25% of thetotal fatty acids in the cell. In a further embodiment, the total fattyacid in the cell has less than 1% C20:1. In preferred embodiments, theextractable TAG in the cell comprises the fatty acids at the levelsreferred to herein. Each possible combination of the features definingthe lipid as described herein is also encompassed.

The level of production of LC-PUFA in the recombinant cell, plant orplant part such as seed may also be expressed as a conversion percentageof a specific substrate fatty acid to one or more product fatty acids,which is also referred to herein as a “conversion efficiency” or“enzymatic efficiency”. This parameter is based on the fatty acidcomposition in the lipid extracted from the cell, plant, plant part orseed, i.e., the amount of the LC-PUFA formed (including other LC-PUFAderived therefrom) as a percentage of one or more substrate fatty acids(including all other fatty acids derived therefrom). The general formulafor a conversion percentage is: 100×(the sum of percentages of theproduct LC-PUFA and all products derived therefrom)/(the sum of thepercentages of the substrate fatty acid and all products derivedtherefrom). With regard to DHA, for example, this may be expressed asthe ratio of the level of DHA (as a percentage in the total fatty acidcontent in the lipid) to the level of a substrate fatty acid (e.g. OA,LA, ALA, SDA, ETA or EPA) and all products other than DHA derived fromthe substrate. The conversion percentage or efficiency of conversion canbe expressed for a single enzymatic step in a pathway, or for part orthe whole of a pathway.

Specific conversion efficiencies are calculated herein according to theformulae:

OA to DHA=100×(% DHA)/(sum % forOA,LA,GLA,DGLA,ARA,EDA,ALA,SDA,ETrA,ETA,EPA,DPA and DHA).  1.

LA to DHA=100×(% DHA)/(sum % forLA,GLA,DGLA,ARA,EDA,ALA,SDA,ETrA,ETA,EPA,DPA and DHA).  2.

ALA to DHA=100×(% DHA)/(sum % for ALA,SDA,ETrA,ETA,EPA,DPA and DHA).  3.

EPA to DHA=100×(% DHA)/(sum % for EPA,DPA and DHA).  4.

DPA to DHA(Δ4-desaturase efficiency)=100×(% DHA)/(sum % for DPA andDHA).  5.

Δ12-desaturase efficiency=100×(sum % forLA,GLA,DGLA,ARA,EDA,ALA,SDA,ETrA,ETA,EPA,DPA and DHA)/(sum % forOA,LA,GLA,DGLA,ARA,EDA,ALA,SDA,ETrA,ETA,EPA,DPA and DHA).  6.

ω3-desaturase efficiency=100×(sum % for ALA,SDA,ETrA,ETA,EPA,DPA andDHA)/(sum % for LA,GLA,DGLA,ARA,EDA,ALA,SDA,ETrA,ETA,EPA,DPA andDHA).  7.

OA to ALA=100×(sum % for ALA,SDA,ETrA,ETA,EPA,DPA and DHA)/(sum % forOA,LA,GLA,DGLA,ARA,EDA,ALA,SDA,ETrA,ETA,EPA,DPA and DHA).  8.

Δ6-desaturase efficiency(on ω3 substrate ALA)=100×(sum % forSDA,ETA,EPA,DPA and DHA)/(% ALA,SDA,ETrA,ETA,EPA,DPA and DHA).  9.

Δ6-elongase efficiency(on ω3 substrate SDA)=100×(sum % for ETA,EPA,DPAand DHA)/(sum % for SDA,ETA,EPA,DPA and DHA).  10.

Δ5-desaturase efficiency(on ω3 substrate ETA)=100×(sum % for EPA,DPA andDHA)/(sum % for ETA,EPA,DPA and DHA).  11.

Δ5-elongase efficiency(on ω3 substrate EPA)=100×(sum % for DPA andDHA)/(sum % for EPA,DPA and DHA).  12.

The fatty acid composition of the lipid, preferably seedoil, of theinvention, is also characterised by the ratio of ω6 fatty acids:ω3 fattyacids in the total fatty acid content, for either total ω6 fattyacids:total ω3 fatty acids or for new ω6 fatty acids:new ω3 fatty acids.The terms total ω6 fatty acids, total ω3 fatty acids, new ω6 fatty acidsand new ω3 fatty acids have the meanings as defined herein. The ratiosare calculated from the fatty acid composition in the lipid extractedfrom the cell, plant, plant part or seed, in the manner as exemplifiedherein. It is desirable to have a greater level of ω3 than ω6 fattyacids in the lipid, and therefore an ω6:ω3 ratio of less than 1.0 ispreferred. A ratio of 0.0 indicates a complete absence of the defined ω6fatty acids; a ratio of 0.03 was achieved as described in Example 6.Such low ratios can be achieved through the combined use of aΔ6-desaturase which has an ω3 substrate preference together with anω3-desaturase, particularly a fungal ω3-desaturase such as the Pichiapastoris ω3-desaturase as exemplified herein.

The yield of LC-PUFA per weight of seed may also be calculated based onthe total oil content in the seed and the % DHA in the oil. For example,if the oil content of canola seed is about 40% (w/w) and about 12% ofthe total fatty acid content of the oil is DHA, the DHA content of theseed is about 4.8% or about 48 mg per gram of seed. As described inExample 2, the DHA content of Arabidopsis seed having about 9% DHA,which has a lower oil content than canola, was about 25 mg/g seed. At aDHA content of about 7%, canola seed or Camelina sativa seed has a DHAcontent of about 28 mg per gram of seed. The present invention thereforeprovides Brassica napus, B. juncea and Camelina sativa plants, and seedobtained therefrom, comprising at least about 28 mg DHA per gram seed.The seed has a moisture content as is standard for harvested mature seedafter drying down (4-15% moisture). The invention also provides aprocess for obtaining oil, comprising obtaining the seed and extractingthe oil from the seed, and uses of the oil and methods of obtaining theseed comprising harvesting the seeds from the plants according to theinvention.

The amount of DHA produced per hectare can also be calculated if theseed yield per hectare is known or can be estimated. For example, canolain Australia typically yields about 2.5 tonnes seed per hectare, whichat 40% oil content yields about 1000 kg of oil. At 12% DHA in the totaloil, this provides about 120 kg of DHA per hectare. If the oil contentis reduced by 50%, this still provides about 60 kg DHA/ha.

Evidence to date suggests that some desaturases expressed heterologouslyin yeast or plants have relatively low activity in combination with someelongases. This may be alleviated by providing a desaturase with thecapacity of to use an acyl-CoA form of the fatty acid as a substrate inLC-PUFA synthesis, and this is thought to be advantageous in recombinantcells particularly in plant cells. A particularly advantageouscombination for efficient DHA synthesis is a fungal ω3-desaturase, forexample such as the Pichia pastoris ω3-desaturase (SEQ ID NO: 12), witha Δ6-desaturase which has a preference for ω3 acyl substrates such as,for example, the Micromonas pusilla Δ6-desaturase (SEQ ID NO: 13), orvariants thereof which have at least 95% amino acid sequence identity.

As used herein, the term “essentially free” means that the composition(for example lipid or oil) comprises little (for example, less thanabout 0.5%, less than about 0.25%, less than about 0.1%, or less thanabout 0.01%) or none of the defined component. In an embodiment,“essentially free” means that the component is undetectable using aroutine analytical technique, for example a specific fatty acid (such asω6-docosapentaenoic acid) cannot be detected using gas chromatography asoutlined in Example 1.

Production of Oils

Techniques that are routinely practiced in the art can be used toextract, process, and analyze the oils produced by cells, plants, seeds,etc of the instant invention. Typically, plant seeds are cooked,pressed, and extracted to produce crude oil, which is then degummed,refined, bleached, and deodorized. Generally, techniques for crushingseed are known in the art. For example, oilseeds can be tempered byspraying them with water to raise the moisture content to, e.g., 8.5%,and flaked using a smooth roller with a gap setting of 0.23 to 0.27 mm.Depending on the type of seed, water may not be added prior to crushing.Application of heat deactivates enzymes, facilitates further cellrupturing, coalesces the oil droplets, and agglomerates proteinparticles, all of which facilitate the extraction process.

In an embodiment, the majority of the seed oil is released by passagethrough a screw press. Cakes expelled from the screw press are thensolvent extracted, e.g., with hexane, using a heat traced column.Alternatively, crude oil produced by the pressing operation can bepassed through a settling tank with a slotted wire drainage top toremove the solids that are expressed with the oil during the pressingoperation. The clarified oil can be passed through a plate and framefilter to remove any remaining fine solid particles. If desired, the oilrecovered from the extraction process can be combined with the clarifiedoil to produce a blended crude oil.

Once the solvent is stripped from the crude oil, the pressed andextracted portions are combined and subjected to normal oil processingprocedures. As used herein, the term “purified” when used in connectionwith lipid or oil of the invention typically means that that theextracted lipid or oil has been subjected to one or more processingsteps of increase the purity of the lipid/oil component. For example, apurification step may comprise one or more or all of the groupconsisting of: degumming, deodorising, decolourising, drying and/orfractionating the extracted oil. However, as used herein, the term“purified” does not include a transesterification process or otherprocess which alters the fatty acid composition of the lipid or oil ofthe invention so as to increase the DHA content as a percentage of thetotal fatty acid content. Expressed in other words, the fatty acidcomposition of the purified lipid or oil is essentially the same as thatof the unpurified lipid or oil.

Degumming

Degumming is an early step in the refining of oils and its primarypurpose is the removal of most of the phospholipids from the oil, whichmay be present as approximately 1-2% of the total extracted lipid.Addition of ˜2% of water, typically containing phosphoric acid, at70-80° C. to the crude oil results in the separation of most of thephospholipids accompanied by trace metals and pigments. The insolublematerial that is removed is mainly a mixture of phospholipids andtriacylglycerols and is also known as lecithin. Degumming can beperformed by addition of concentrated phosphoric acid to the crudeseedoil to convert non-hydratable phosphatides to a hydratable form, andto chelate minor metals that are present. Gum is separated from theseedoil by centrifugation.

Alkali Refining

Alkali refining is one of the refining processes for treating crude oil,sometimes also referred to as neutralization. It usually followsdegumming and precedes bleaching. Following degumming, the seedoil cantreated by the addition of a sufficient amount of an alkali solution totitrate all of the fatty acids and phosphoric acids, and removing thesoaps thus formed. Suitable alkaline materials include sodium hydroxide,potassium hydroxide, sodium carbonate, lithium hydroxide, calciumhydroxide, calcium carbonate and ammonium hydroxide. This process istypically carried out at room temperature and removes the free fattyacid fraction. Soap is removed by centrifugation or by extraction into asolvent for the soap, and the neutralised oil is washed with water. Ifrequired, any excess alkali in the oil may be neutralized with asuitable acid such as hydrochloric acid or sulphuric acid.

Bleaching

Bleaching is a refining process in which oils are heated at 90-120° C.for 10-30 minutes in the presence of a bleaching earth (0.2-2.0%) and inthe absence of oxygen by operating with nitrogen or steam or in avacuum. This step in oil processing is designed to remove unwantedpigments (carotenoids, chlorophyll, gossypol etc), and the process alsoremoves oxidation products, trace metals, sulphur compounds and tracesof soap.

Deodorization

Deodorization is a treatment of oils and fats at a high temperature(200-260° C.) and low pressure (0.1-1 mm Hg). This is typically achievedby introducing steam into the seedoil at a rate of about 0.1ml/minute/100 ml of seedoil. After about 30 minutes of sparging, theseedoil is allowed to cool under vacuum. The seedoil is typicallytransferred to a glass container and flushed with argon before beingstored under refrigeration. This treatment improves the colour of theseedoil and removes a majority of the volatile substances or odorouscompounds including any remaining free fatty acids, monoacylglycerolsand oxidation products.

Winterisation

Winterization is a process sometimes used in commercial production ofoils for the separation of oils and fats into solid (stearin) and liquid(olein) fractions by crystallization at sub-ambient temperatures. It wasapplied originally to cottonseed oil to produce a solid-free product. Itis typically used to decrease the saturated fatty acid content of oils.

Transesterification

Transesterification is a process that exchanges the fatty acids withinand between TAGs or transfers the fatty acids to another alcohol to forman ester, initially by releasing fatty acids from the TAGs either asfree fatty acids or as fatty acid esters, usually fatty acid methylesters or ethyl esters. When combined with a fractionation process,transesterification can be used to modify the fatty acid composition oflipids (Marangoni et al., 1995). Transesterification can use eitherchemical (e.g. strong acid or base catalysed) or enzymatic means, thelatter using lipases which may be position-specific (sn-1/3 or sn-2specific) for the fatty acid on the TAG, or having a preference for somefatty acids over others (Speranza et al, 2012). The fatty acidfractionation to increase the concentration of LC-PUFA in an oil can beachieved by any of the methods known in the art, such as, for example,freezing crystallization, complex formation using urea, moleculardistillation, supercritical fluid extraction and silver ion complexing.Complex formation with urea is a preferred method for its simplicity andefficiency in reducing the level of saturated and monounsaturated fattyacids in the oil (Gamez et al., 2003). Initially, the TAGs of the oilare split into their constituent fatty acids, often in the form of fattyacid esters, by hydrolysis under either acid or base catalysed reactionconditions, whereby one mol of TAG is reacted with at least 3 mol ofalcohol (e.g. ethanol for ethyl esters or methanol for methyl esters)with excess alcohol used to enable separation of the formed alkyl estersand the glycerol that is also formed, or by lipases. These free fattyacids or fatty acid esters, which are usually unaltered in fatty acidcomposition by the treatment, may then be mixed with an ethanolicsolution of urea for complex formation. The saturated andmonounsaturated fatty acids easily complex with urea and crystallize outon cooling and may subsequently be removed by filtration. The non-ureacomplexed fraction is thereby enriched with LC-PUFA.

Feedstuffs

The present invention includes compositions which can be used asfeedstuffs. For purposes of the present invention, “feedstuffs” includeany food or preparation for human or animal consumption which when takeninto the body (a) serve to nourish or build up tissues or supply energy;and/or (b) maintain, restore or support adequate nutritional status ormetabolic function. Feedstuffs of the invention include nutritionalcompositions for babies and/or young children such as, for example,infant formula, and seedmeal of the invention.

Feedstuffs of the invention comprise, for example, a cell of theinvention, a plant of the invention, the plant part of the invention,the seed of the invention, an extract of the invention, the product ofthe method of the invention, the product of the fermentation process ofthe invention, or a composition along with a suitable carrier(s). Theterm “carrier” is used in its broadest sense to encompass any componentwhich may or may not have nutritional value. As the skilled addresseewill appreciate, the carrier must be suitable for use (or used in asufficiently low concentration) in a feedstuff such that it does nothave deleterious effect on an organism which consumes the feedstuff

The feedstuff of the present invention comprises an oil, fatty acidester, or fatty acid produced directly or indirectly by use of themethods, cells or plants disclosed herein. The composition may either bein a solid or liquid form. Additionally, the composition may includeedible macronutrients, protein, carbohydrate, vitamins, and/or mineralsin amounts desired for a particular use. The amounts of theseingredients will vary depending on whether the composition is intendedfor use with normal individuals or for use with individuals havingspecialized needs, such as individuals suffering from metabolicdisorders and the like.

Examples of suitable carriers with nutritional value include, but arenot limited to, macronutrients such as edible fats, carbohydrates andproteins. Examples of such edible fats include, but are not limited to,coconut oil, borage oil, fungal oil, black current oil, soy oil, andmono- and diglycerides. Examples of such carbohydrates include (but arenot limited to): glucose, edible lactose, and hydrolyzed starch.Additionally, examples of proteins which may be utilized in thenutritional composition of the invention include (but are not limitedto) soy proteins, electrodialysed whey, electrodialysed skim milk, milkwhey, or the hydrolysates of these proteins.

With respect to vitamins and minerals, the following may be added to thefeedstuff compositions of the present invention: calcium, phosphorus,potassium, sodium, chloride, magnesium, manganese, iron, copper, zinc,selenium, iodine, and Vitamins A, E, D, C, and the B complex. Other suchvitamins and minerals may also be added.

The components utilized in the feedstuff compositions of the presentinvention can be of semi-purified or purified origin. By semi-purifiedor purified is meant a material which has been prepared by purificationof a natural material or by de novo synthesis.

A feedstuff composition of the present invention may also be added tofood even when supplementation of the diet is not required. For example,the composition may be added to food of any type, including (but notlimited to): margarine, modified butter, cheeses, milk, yogurt,chocolate, candy, snacks, salad oils, cooking oils, cooking fats, meats,fish and beverages.

The genus Saccharomyces spp is used in both brewing of beer and winemaking and also as an agent in baking, particularly bread. Other yeastssuch as oleaginous yeast including, for example, Yarrowia spp, are alsouseful in LC-PUFA production. Yeasts may be used as an additive inanimal feed, such as in aquaculture. It will be apparent thatgenetically engineered yeast strains can be provided which are adaptedto synthesise LC-PUFA as described herein. These yeast strains, orLC-PUFA produced therein, can then be used in food stuffs and in wineand beer making to provide products which have enhanced fatty acidcontent.

Additionally, fatty acids produced in accordance with the presentinvention or host cells transformed to contain and express the subjectgenes may also be used as animal food supplements to alter an animal'stissue, egg or milk fatty acid composition to one more desirable forhuman or animal consumption. Examples of such animals include sheep,cattle, horses, poultry such as chickens and the like.

Furthermore, feedstuffs of the invention can be used in aquaculture toincrease the levels of fatty acids in fish or crustaceans such as, forexample, prawns for human or animal consumption. Preferred fish aresalmon.

Preferred feedstuffs of the invention are the plants, seed and otherplant parts such as leaves and stems which may be used directly as foodor feed for humans or other animals. For example, animals may grazedirectly on such plants grown in the field or be fed more measuredamounts in controlled feeding. The invention includes the use of suchplants and plant parts as feed for increasing the LC-PUFA levels inhumans and other animals.

Compositions

The present invention also encompasses compositions, particularlypharmaceutical compositions, comprising one or more of the fatty acidsand/or resulting oils produced using the methods of the invention.

A pharmaceutical composition may comprise one or more of the fatty acidsand/or oils, in combination with a standard, well-known, non-toxicpharmaceutically-acceptable carrier, adjuvant or vehicle such asphosphate-buffered saline, water, ethanol, polyols, vegetable oils, awetting agent or an emulsion such as a water/oil emulsion. Thecomposition may be in either a liquid or solid form. For example, thecomposition may be in the form of a tablet, capsule, ingestible liquidor powder, indictable, or topical ointment or cream. Proper fluidity canbe maintained, for example, by the maintenance of the required particlesize in the case of dispersions and by the use of surfactants. It mayalso be desirable to include isotonic agents, for example, sugars,sodium chloride, and the like. Besides such inert diluents, thecomposition can also include adjuvants, such as wetting agents,emulsifying and suspending agents, sweetening agents, flavoring agentsand perfuming agents.

Suspensions, in addition to the active compounds, may comprisesuspending agents such as ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanthor mixtures of these substances.

Solid dosage forms such as tablets and capsules can be prepared usingtechniques well known in the art. For example, fatty acids produced inaccordance with the present invention can be tableted with conventionaltablet bases such as lactose, sucrose, and cornstarch in combinationwith binders such as acacia, cornstarch or gelatin, disintegratingagents such as potato starch or alginic acid, and a lubricant such asstearic acid or magnesium stearate. Capsules can be prepared byincorporating these excipients into a gelatin capsule along withantioxidants and the relevant fatty acid(s).

For intravenous administration, the fatty acids produced in accordancewith the present invention or derivatives thereof may be incorporatedinto commercial formulations.

A typical dosage of a particular fatty acid is from 0.1 mg to 20 g,taken from one to five times per day (up to 100 g daily) and ispreferably in the range of from about 10 mg to about 1, 2, 5, or 10 gdaily (taken in one or multiple doses). As known in the art, a minimumof about 300 mg/day of fatty acid, especially LC-PUFA, is desirable.However, it will be appreciated that any amount of fatty acid will bebeneficial to the subject.

Possible routes of administration of the pharmaceutical compositions ofthe present invention include, for example, enteral (e.g., oral andrectal) and parenteral. For example, a liquid preparation may beadministered orally or rectally. Additionally, a homogenous mixture canbe completely dispersed in water, admixed under sterile conditions withphysiologically acceptable diluents, preservatives, buffers orpropellants to form a spray or inhalant.

The dosage of the composition to be administered to the patient may bedetermined by one of ordinary skill in the art and depends upon variousfactors such as weight of the patient, age of the patient, overallhealth of the patient, past history of the patient, immune status of thepatient, etc.

Additionally, the compositions of the present invention may be utilizedfor cosmetic purposes. It may be added to pre-existing cosmeticcompositions such that a mixture is formed or a fatty acid producedaccording to the subject invention may be used as the sole “active”ingredient in a cosmetic composition.

EXAMPLES Example 1. Materials and Methods Expression of Genes in PlantCells in a Transient Expression System

Exogenous genetic constructs were expressed in plant cells in atransient expression system essentially as described by Voinnet et al.(2003) and Wood et al. (2009). Plasmids containing a coding region to beexpressed from a strong constitutive promoter such as the CaMV 35Spromoter were introduced into Agrobacterium tumefaciens strain AGL1. Achimeric gene 35S:p19 for expression of the p19 viral silencingsuppressor was separately introduced into AGL1, as described in WO2010/057246. The recombinant Agrobacterium cells were grown at 28° C. inLB broth supplemented with 50 mg/L kanamycin and 50 mg/L rifampicin tostationary phase. The bacteria were then pelleted by centrifugation at5000 g for 15 min at room temperature before being resuspended toOD600=1.0 in an infiltration buffer containing 10 mM MES pH 5.7, 10 mMMgCl₂ and 100 μM acetosyringone. The cells were then incubated at 28° C.with shaking for 3 hours before equal volumes of Agrobacterium culturescontaining 35S:p19 and the test chimeric construct(s) of interest weremixed prior to infiltration into leaf tissue. The plants were typicallygrown for a further five days after infiltration before leaf discs weretaken and freeze-dried for GC analysis of the fatty acids.

Fatty acid methyl esters (FAME) of total leaf lipids in freeze-driedsamples were produced by incubating the samples inmethanol/HCl/dichloromethane (10/1/1 v/v) solution for 2 hours at 80° C.together with a known amount of hexadecanoic acid as an internalstandard. FAMEs were extracted in hexane/DCM, concentrated to a smallvolume in hexane and injected into a GC. The amount of individual andtotal fatty acids present in the lipid fractions were quantified on thebasis of the known amount of internal standard.

Gas Chromatography (GC) Analysis of Fatty Acids

FAME were analysed by gas chromatography using an Agilent Technologies7890A GC (Palo Alto, Calif., USA) equipped with a 30 m SGE-BPX70 column(70% cyanopropyl polysilphenylene-siloxane, 0.25 mm inner diameter, 0.25mm film thickness), an FID, a split/splitless injector and an AgilentTechnologies 7693 Series auto sampler and injector. Helium was used asthe carrier gas. Samples were injected in split mode (50:1 ratio) at anoven temperature of 150° C. After injection, the oven temperature washeld at 150° C. for 1 min then raised to 210° C. at 3° C. min⁻¹, againraised to 240° C. at 50° C. min⁻¹ and finally holding for 1.4 min at240° C. Peaks were quantified with Agilent Technologies ChemStationsoftware (Rev B.04.03 (16), Palo Alto, Calif., USA) based on theresponse of the known amount of the external standard GLC-411 (Nucheck)and C17:0-ME internal standard.

Liquid Chromatography-Mass Spectrometry (LC-MS) Analysis of Lipids

Total lipids were extracted from freeze-dried developing seeds, twelvedays after flowering (daf), and mature seeds after adding a known amountof tri-C17:0-TAG as an internal quantitation standard. The extractedlipids were dissolved into 1 mL of 10 mM butylated hydroxytoluene inbutanol:methanol (1:1 v/v) per 5 mg dry material and analysed using anAgilent 1200 series LC and 6410b electrospray ionisation triplequadrupole LC-MS. Lipids were chromatographically separated using anAscentis Express RP-Amide column (50 mm×2.1 mm, 2.7 μm, Supelco)operating a binary gradient with a flow rate of 0.2 mL/min. The mobilephases were: A. 10 mM ammonium formate in H₂O:methanol: tetrahydrofuran(50:20:30 v/v/v); B. 10 mM ammonium formate in H₂O:methanol:tetrahydrofuran (5:20:75, v/v/v). Multiple reaction monitoring (MRM)lists were based on the following major fatty acids: 16:0, 18:0, 18:1,18:2, 18:3, 18:4, 20:1, 20:2, 20:3, 20:4, 20:5, 22:4, 22:5, 22:6 using acollision energy of 30 V and fragmentor of 60 V. Individual MRM TAG wasidentified based on ammoniated precursor ion and product ion fromneutral loss of 22:6. TAG was quantified using a 10 μM tristearinexternal standard.

Determination of Seed Fatty Acid Profile and Oil Content

Where seed oil content was to be determined, seeds were dried in adesiccator for 24 h and approximately 4 mg of seed was transferred to a2 ml glass vial containing Teflon-lined screw cap. 0.05 mgtriheptadecanoin dissolved in 0.1 ml toluene was added to the vial asinternal standard.

Seed FAME were prepared by adding 0.7 ml of 1N methanolic HCl (Supelco)to the vial containing seed material, vortexed briefly and incubated at80° C. for 2 h. After cooling to room temperature, 0.3 ml of 0.9% NaCl(w/v) and 0.1 ml hexane was added to the vial and mixed well for 10 minin Heidolph Vibramax 110. The FAME was collected into 0.3 ml glassinsert and analysed by GC with a flame ionization detector (FID) asmentioned earlier.

The peak area of individual FAME were first corrected on the basis ofthe peak area responses of known amount of the same FAMEs present in acommercial standard GLC-411 (NU-CHEK PREP, INC., USA). GLC-411 containsequal amounts of 31 fatty acids (% by wt), ranging from C8:0 to C22:6.In case of fatty acids, which were not present in the standard, theinventors took the peak area responses of the most similar FAME. Forexample, peak area response of FAMEs of 16:1d9 was used for 16:1d7 andFAME response of C22:6 was used for C22:5. The corrected areas were usedto calculate the mass of each FAME in the sample by comparison to theinternal standard mass. Oil is stored mainly in the form of TAG and itsweight was calculated based on FAME weight. Total moles of glycerol wasdetermined by calculating moles of each FAMES and dividing total molesof FAMEs by three. TAG was calculated as the sum of glycerol and fattyacyl moieties using a relation: % oil by weight=100×((41× total molFAME/3)+(total g FAME−(15× total mol FAME)))/g seed, where 41 and 15 aremolecular weights of glycerol moiety and methyl group, respectively.

Analysis of the Sterol Content of Oil Samples

Samples of approximately 10 mg of oil together with an added aliquot ofC24:0 monol as an internal standard were saponified using 4 mL 5% KOH in80% MeOH and heating for 2 h at 80° C. in a Teflon-lined screw-cappedglass tube. After the reaction mixture was cooled, 2 mL of Milli-Q waterwere added and the sterols were extracted into 2 mL of hexane:dichloromethane (4:1 v/v) by shaking and vortexing. The mixture wascentrifuged and the sterol extract was removed and washed with 2 mL ofMilli-Q water. The sterol extract was then removed after shaking andcentrifugation. The extract was evaporated using a stream of nitrogengas and the sterols silylated using 200 mL of BSTFA and heating for 2 hat 80° C.

For GC/GC-MS analysis of the sterols, sterol-OTMSi derivatives weredried under a stream of nitrogen gas on a heat block at 40° C. and thenre-dissolved in chloroform or hexane immediately prior to GC/GC-MSanalysis. The sterol-OTMS derivatives were analysed by gaschromatography (GC) using an Agilent Technologies 6890A GC (Palo Alto,Calif., USA) fitted with an Supelco Equity™-1 fused silica capillarycolumn (15 m×0.1 mm i.d., 0.1 μm film thickness), an FID, asplit/splitless injector and an Agilent Technologies 7683B Series autosampler and injector. Helium was the carrier gas. Samples were injectedin splitless mode at an oven temperature of 120° C. After injection, theoven temperature was raised to 270° C. at 10° C. min⁻¹ and finally to300° C. at 5° C. min¹. Peaks were quantified with Agilent TechnologiesChemStation software (Palo Alto, Calif., USA). GC results are subject toan error of +5% of individual component areas.

GC-mass spectrometric (GC-MS) analyses were performed on a FinniganThermoquest GCQ GC-MS and a Finnigan Thermo Electron Corporation GC-MS;both systems were fitted with an on-column injector and ThermoquestXcalibur software (Austin, Tex., USA). Each GC was fitted with acapillary column of similar polarity to that described above. Individualcomponents were identified using mass spectral data and by comparingretention time data with those obtained for authentic and laboratorystandards. A full procedural blank analysis was performed concurrent tothe sample batch.

RT-PCR Conditions

Reverse transcription-PCR (RT-PCR) amplification was typically carriedout using the Superscript III One-Step RT-PCR system (Invitrogen) in avolume of 25 μL using 10 pmol of the forward primer and 30 pmol of thereverse primer, MgSO₄ to a final concentration of 2.5 mM, 400 ng oftotal RNA with buffer and nucleotide components according to themanufacturer's instructions. Typical temperature regimes were: 1 cycleof 45° C. for 30 minutes for the reverse transcription to occur; then 1cycle of 94° C. for 2 minutes followed by 40 cycles of 94° C. for 30seconds, 52° C. for 30 seconds, 70° C. for 1 minute; then 1 cycle of 72°C. for 2 minutes before cooling the reaction mixtures to 5° C.

Production of B. napus Somatic Embryos by Induction with 35S-LEC2

B. napus (cv. Oscar) seeds were sterilized using chlorine gas asdescribed by (Attila Kereszt et al., 2007). Sterilized seeds weregerminated on ½ strength MS media (Murashige and Skoog, 1962) with 0.8%agar adjusted to pH 5.8, and grown at 24° C. under fluorescent lighting(50 μE/m²s) with a 18/6 h (light/dark) photoperiod for 6-7 days.Cotyledonary petioles with 2-4 mm stalk length were isolated asepticallyfrom these seedlings and used as explants. Cultures of the transformedA. tumefaciens strain AGL1, one harbouring a seed specific binary vectorand a second with a 35S-LEC2 construct were inoculated from singlecolonies from fresh plates and grown in 10 mL of LB medium withappropriate antibiotics and grown overnight at 28° C. with agitation at150 rpm. The bacterial cells were collected by centrifugation at 4000rpm for 5 minutes, washed with MS media containing 2% sucrose andre-suspended in 10 mL of the same medium and grown with antibiotics forselection as appropriate for 4 hours after the addition ofacetosyringone to 100 μM. Two hours before addition to the planttissues, spermidine was added to a final concentration of 1.5 mM and thefinal density of the bacteria adjusted to OD 600 nm=0.4 with freshmedium. The two bacterial cultures, one carrying the seed specificconstruct and other carrying 35S-AtLEC2, were mixed in 1:1 to 1:1.5ratios.

Freshly-isolated B. napus cotyledonary petioles were infected with 20 mLA. tumefaciens cultures for 6 minutes. The cotyledonary petioles wereblotted on sterile filter paper to remove excess A. tumefaciens and thentransferred to co-cultivation media (MS media with 1 mg/L TDZ, 0.1 mg/LNAA, 100 μM acetosyringone supplemented with L-cysteine (50 mg/L),ascorbic acid (15 mg/L) and MES (250 mg/1)). The plates were sealed withmicro-pore tape and incubated in the dark at 24° C. for 48 hrs. Theco-cultivated explants were transferred to pre-selection media (MScontaining 1 mg/L TDZ, 0.1 mg/L NAA, 3 mg/L AgNO₃, 250 mg/L cefotaximeand 50 mg/L timentin) and cultured for 4-5 days at 24° C. with a 16 h/8h photoperiod. The explants were then transferred to selection media (MScontaining 1 mg/L TDZ, 0.1 mg/L NAA, 3 mg/L AgNO₃, 250 mg/L cefotaximeand 50 mg/L timentin) according to the selectable marker gene on theseed specific vector and cultured for 2-3 weeks at 24° C. with a 16 h/8h photoperiod. Explants with green embryogenic callus were transferredto hormone free MS media (MS with 3 mg/L AgNO₃, 250 mg/L cefotaxime, 50mg/L timentin and the selection agent) and cultured for another 2-3weeks. Torpedo or cotyledonary stage embryos isolated from survivingexplants on the selection medium were analysed for fatty acidcomposition in their total lipid using GC.

Example 2. Stable Expression of Transgenic DHA Pathways in Arabidopsisthaliana Seeds Binary Vector Construction

The binary vectors pJP3416-GA7 and pJP3404 each contained sevenheterologous fatty acid biosynthesis genes, encoding 5 desaturases and 2elongases, and a plant selectable marker between the left and rightborder repeats of the T-DNA present in each vector (FIGS. 2 and 3). SEQID NO:1 provides the nucleotide sequence of the T-DNA region ofpJP3416-GA7 from the right to left border sequences. Both geneticconstructs contained plant codon-optimised genes encoding a Lachanceakluyveri Δ12-desaturase (comprising nucleotides 14143-16648 of SEQ IDNO:1), a Pichia pastoris ω3-desaturase (comprising nucleotides7654-10156 of SEQ ID NO:1), a Micromonas pusilla Δ6-desaturase(comprising nucleotides 226-2309 of SEQ ID NO: 1), Pavlova salina Δ5-and Δ4-desaturases (comprising nucleotides 4524-6485 and 10157-14142 ofSEQ ID NO:1, respectively) and Pyramimonas cordata Δ6- and Δ5-elongases(comprising nucleotides 2310-4523 and 17825-19967 of SEQ ID NO:1,respectively). The specific regions of the T-DNA (Orientation: right toleft border sequences) region of the binary vector pJP3416-GA7 withrespect to SEQ ID NO: 1 are as follows:

Nucleotides 1-163: Right border; 480-226, Agrobacterium tumefaciensnopaline synthase terminator (TER NOS); 1883-489, Micromonas pusillaΔ6-desaturase; 2309-1952, Brassica napus truncated napin promoter (PROFP1); 2310-3243, Arabidopsis thaliana FAE1 promoter (PRO_FAE1);3312-4181, Pyramimonas cordata Δ6-elongase; 4190-4523, Glycine maxlectin terminator (TER Lectin); 4524-4881, PRO FP1; 4950-6230: Pavlovasalina Δ5-desaturase; 6231-6485: TER NOS; 7653-6486, Nicotiana tabacumRb7 matrix attachment region (MAR); 8387-7654, Linum usitatissimumconlinin1 terminator (TER_Cnl1); 9638-8388, Pichia pastorisω3-desaturase; 10156-9707, Linum usitatissimum conlinin1 promoter(PRO_Cnl1); 10157-12189, Linum usitatissimum conlinin1 promoter;12258-13604, Pavlova salina Δ4-desaturase; 13605-14142, Linumusitatissimum conlinin2 terminator; 14143-14592, PRO_Cnl1; 14661-15914,Lachancea kluyveri Δ12-desaturase; 15915-16648, TER_Cnl1; 17816-16649,MAR; 17825-18758, PRO_FAE1; 18827-19633, Pyramimonas cordataΔ5-elongase; 19634-19967, TER_Lectin; 19990-20527, Cauliflower mosaicvirus 35S promoter with duplicated enhancer region; 20537-21088,Streptomyces viridochromogenes phosphinothricin-N-acetyltransferase;21097-21349, TER NOS; 21367-21527, Left border.

The seven coding regions in the constructs were each under the controlof a seed specific promoter—three different promoters were used, namelythe truncated Brassica napus napin promoter (pBnFP1), the Arabidopsisthaliana FAE1 promoter (pAtFAE1) and the Linum usitatissimum conlinin 1promoter (pLuCnl1). The seven fatty acid biosynthesis genes togethercoded for an entire DHA synthesis pathway that was designed to convert18:1^(Δ9) (oleic acid) through to 22:6^(Δ4,7,10,13,16,19) (DHA). Bothbinary vectors contained a BAR plant selectable marker coding regionoperably linked to a Cauliflower Mosaic Virus (CaMV) 35S promoter withduplicated enhancer region and A. tumefaciens nos3′ polyadenylationregion-transcription terminator. The plant selectable marker wassituated adjacent to the left border of the T-DNA region, thereforedistally located on the T-DNA with respect to the orientation of T-DNAtransfer into the plant cells. This increased the likelihood thatpartial transfer of the T-DNA, which would be likely to not include theselectable marker gene, would not be selected. pJP3416-GA7 and pJP3404each contained an RiA4 origin of replication from Agrobacteriumrhizogenes (Hamilton, 1997).

pJP3416-GA7 was generated by synthesising the DNA region correspondingto nucleotides 226-19975 of SEQ ID NO: 1 (GA7 region) and inserting thisregion into the recipient binary vector pJP3416 at the PspOMI site. Eachfatty acid biosynthetic gene on GA7 included a Tobacco Mosaic Virus 5′untranslated region (5′UTR) sequence which was operably linked to eachcoding region, between the promoter and the translation initiation ATG,to maximise translation efficiency of the mRNAs produced from the genes.The GA7 construct also included two Nicotiana tabacum Rb7 matrixattachment region (MAR) sequences, as described by Hall et al. (1991).MAR sequences, sometimes termed nuclear attachment regions, are known tobind specifically to the nuclear matrix in vitro and may mediate bindingof chromatin to the nuclear matrix in vivo. MARs are thought to functionto reduce transgene silencing. In pJP3416-GA7 the MARs were alsoinserted and positioned within the T-DNA region in order to act as DNAspacers to insulate transgenic expression cassettes. The pJP3416 vectorprior to insertion of the GA7 region contained only the plant selectablemarker cassette between the borders.

The genetic construct pJP3404 was made by sequential restrictionenzyme-based insertions in which gene cassettes were added to the binaryvector, pJP3367, which comprised genes for production of SDA in seeds.This construct contained genes encoding the L. kluyveri Δ12-desaturaseand P. pastoris ω3-desaturase, both expressed by the B. napus truncatednapin promoter (FP1), and the M. pusilla Δ6-desaturase expressed by theA. thaliana FAE1 promoter (FIG. 4). First, the A. thaliana FAD2 intronwas flanked by EcoRI sites and cloned into the pJP3367 MfeI site togenerate pJP3395. A fragment containing the P. cordata Δ6- andΔ5-elongase cassettes driven by the FAE1 and FP1 promoters,respectively, was cloned into the KasI site of pJP3395 to generatepJP3398. pJP3399 was then generated by replacing the RK2 origin ofreplication in pJP3398 with a RiA4 origin of replication. The finalbinary vector, pJP3404, was generated by cloning a SbfI-flanked fragmentcontaining the P. salina Δ5- and Δ4-desaturase cassettes driven by theFP1 and FAE1 promoters, respectively, into the SbfI site of pJP3399.

A. thaliana Transformation and Analysis of Fatty Acid Composition

The chimeric vectors were introduced into A. tumefaciens strain AGL1 andcells from cultures of the transformed Agrobacterium used to treat A.thaliana (ecotypes Columbia and a fad2 mutant) plants using the floraldip method for transformation (Clough and Bent, 1998). After maturation,the T₁ seeds from the treated plants were harvested and plated onto MSplates containing PPT for selection of plants containing the BARselectable marker gene. Surviving, healthy T₁ seedlings were transferredto soil. After growth of the plants to maturity and allowing forself-fertilisation, T₂ seeds from these plants were harvested and thefatty acid composition of their seed lipid analysed by GC analysis asdescribed in Example 1.

The data for the DHA level in the seed lipids are shown in FIG. 5 (laneslabelled T₂) for 13 transformants using pJP3416-GA7 into the Columbiagenetic background, and for six transformants using the fad2 mutant. ThepJP3416-GA7 construct resulted in the production of slightly higherlevels of DHA, as a percentage of total fatty acid content, on averagethan the pJP3404 construct. Table 4 shows the fatty acid composition oftotal seed lipid from the T₂ lines with the highest DHA levels. Thecalculated conversion efficiencies for each enzymatic step in theproduction of DHA from oleic acid in the same seeds are shown in Table5. Conversion efficiencies were calculated as (% products×100)/(%remaining substrate+% products), thereby expressed as a percentage.

The highest observed level of DHA produced in the pJP3416-GA7 T₂transformed lines was 6.2%, additionally with 0.5% EPA and 0.2% DPA(line #14). These T₂ seeds were still segregating for the transgene i.e.were not yet uniformly homozygous. Compiled data from the total seedlipid profiles from independent transgenic seed (Table 4) are shown inTable 6. The level of ω3 fatty acids produced as a result of thetransgenes in these seeds (total new ω3 fatty acids, excluding the levelof ALA which was produced endogenously in the Columbia background) was10.7% while the level of ω6 fatty acids (total new ω6 fatty acids butexcluding 18:2^(Δ9,12)) was 1.5%. This represents an extremelyfavourable ration of new ω3 fatty acids:new ω6 fatty acids, namely7.3:1.

T₂ seeds of selected lines transformed with pJP3416-GA7, namely forlines designated 7, 10, 14, 22 and 34 in the Columbia background and forlines designated 18, 21 and 25 in the fad2 mutant background, wereplated onto MS media containing PPT for selection of transgenicseedlings in vitro. Twenty PPT-resistant seedlings for each line weretransferred to soil and grown to maturity after self-fertilisation.These plants were highly likely to be homozygous for the selectablemarker gene, and therefore for at least one T-DNA insertion in thegenome of the plants. T₃ seed from these plants were harvested andanalysed for fatty acid composition in their seedoil by GC. The data areshown in Table 7. This analysis revealed that the pJP3416-GA7 constructgenerated higher levels of the ω3 LC-PUFA DHA in T₃ seeds of thehomozygous plants than in the segregating T₂ seed. Up to about 13.9% DHAwas observed in the T₃ pJP3416-GA7 transformed line designated 22.2 inthe Columbia background, increased from about 5.5% in the hemizygous T₂seed, with a sum level of about 24.3% of new ω3 fatty acids as apercentage of the total fatty acids in the seed lipid content. New ω6fatty acids were at a level of 1.1% of total fatty acids, representing avery favourable ratio of new ω3 fatty acids:new ω6 fatty acids, namelyabout 22:1. Similarly, transformants in the fad2 mutant backgroundyielded 20.6% as a sum of new ω3 fatty acids, including 11.5% DHA, as apercentage of the total fatty acids in the seed lipid content.

TABLE 4 Fatty acid composition of total seed lipid from independenttransgenic T₂ Arabidopsis seeds with DHA levels at the higher end of theobserved range. ‘Col’ refers to the Columbia ecotype and ‘FAD2’ to thefad2 mutant ecotype. ‘GA7’ refers to transformation with the T-DNA ofthe pJP3416-GA7 vector, pJP3404 with the T-DNA of the pJP3404 vector.20:1n-9 and 20:1n-11 fatty acids were not resolved in the GC analysis.“Other minor” fatty acids include 14:0, 16:1n7, 16:1n9, 16:1n13t,16:2n6, 16:3n3, i18:0, 18:1n5, 20:1n5, 22:0, 22:1n7, 22:1n11/n13, 24:0,24:1n9. pJP3404_Col_#1 pJP3404_FAD2_#31 GA7_Col_#7 GA7_Col_#34GA7_Col_#2 GA7_Col_#10 16:0 9.6 7.8 8.7 8.2 8.7 8.6 18:0 2.9 3.9 3.7 3.93.6 3.3 18:1d11 2.2 1.8 2.0 1.9 2.0 2.3 20:0 1.6 2.3 2.0 2.0 2.1 1.620:1d13 2.2 1.8 1.6 1.5 1.7 1.6 20:1d9/d11 13.0 15.9 16.1 16.1 16.3 15.022:1d13 1.1 1.2 1.1 1.1 1.3 1.0 Other minor 1.9 1.5 1.5 1.4 1.5 1.318:1d9 10.8 14.0 10.6 10.6 10.1 11.1 18:2ω6 28.9 28.3 16.4 16.1 18.213.7 18:3ω3 16.6 14.9 29.6 29.6 27.5 32.4 18:3ω6 0.7 0.5 0.1 0.1 0.1 0.120:2ω6 1.6 1.5 1.1 1.2 1.3 1.0 20:3ω6 0.0 0.0 0.0 0.0 0.0 0.0 20:4ω6 0.00.0 0.0 0.0 0.0 0.0 22:4ω6 1.6 0.6 0.3 0.3 0.3 0.4 22:5ω6 0.1 0.1 0.00.0 0.0 0.0 18:4ω3 1.0 0.5 1.2 1.1 1.1 1.5 20:3ω3 0.0 0.0 0.0 0.6 0.00.0 20:4ω3 0.4 0.6 0.6 0.7 0.5 0.8 20:5ω3 0.2 0.2 0.3 0.3 0.3 0.3 22:5ω30.0 0.2 0.2 0.2 0.2 0.2 22:6ω3 3.6 2.4 3.0 3.1 3.3 3.9 GA7_Col_#22GA7_Col_#14 GA7_FAD2_#25 GA7_FAD2_#21 GA7_FAD2_#18 16:0 8.3 9.7 7.2 8.57.5 18:0 3.4 3.6 3.2 3.9 3.0 18:1d11 2.3 2.7 1.9 2.0 1.8 20:0 1.6 1.81.6 2.2 1.5 20:1d13 1.5 1.7 1.5 1.7 1.4 20:1d9/d11 13.9 13.5 18.3 15.917.0 22:1d13 1.0 1.0 1.0 1.3 1.2 Other minor 1.6 1.7 1.6 1.4 1.6 18:1d910.0 7.7 26.0 8.2 20.9 18:2ω6 13.7 11.4 6.6 16.6 4.3 18:3ω3 30.4 32.821.9 27.7 30.1 18:3ω6 0.2 0.1 0.1 0.2 0.1 20:2ω6 1.0 1.0 0.4 1.4 0.420:3ω6 0.0 0.0 0.0 0.0 0.0 20:4ω6 0.0 0.0 0.0 0.0 0.0 22:4ω6 0.5 0.4 0.50.4 0.4 22:5ω6 0.0 0.0 0.0 0.0 0.0 18:4ω3 2.7 2.7 1.9 1.8 1.7 20:3ω3 0.60.7 0.0 0.8 0.6 20:4ω3 0.8 0.4 1.0 0.8 0.8 20:5ω3 0.7 0.5 0.6 0.4 0.522:5ω3 0.2 0.2 0.3 0.3 0.3 22:6ω3 5.5 6.2 4.3 4.4 4.8

TABLE 5 Conversion efficiencies of the individual enzymatic steps forproduction of DHA from oleic acid, observed in total seed lipid fromindependent transgenic seed as for Table 4. pJP3404_Col_#1pJP3404_FAD2_#31 GA7_Col_#7 GA7_Col_#34 GA7_Col_#2 GA7_Col_#10 d12-des69.6% 62.5% 66.4% 66.6% 66.7% 67.5% d15-des 39.8% 37.8% 66.1% 66.8%62.3% 72.1% Omega-6 d6-des 4.5% 2.5% 0.7% 0.7% 0.7% 0.9% (d9-elo) 3.1%3.1% 2.2% 2.3% 2.4% 1.8% d6-elo 71.4% 56.9% 83.3% 83.4% 83.0% 84.7%d5-des 100.0% 100.0% 100.0% 100.0% 100.0% 100.0% d5-elo 100.0% 97.8%100.0% 100.0% 100.0% 100.0% d4-des 6.2% 13.0% 0.0% 0.0% 0.0% 0.0%Omega-3 d6-des 23.9% 21.0% 15.2% 15.4% 16.4% 17.1% (d9-elo) 0.0% 0.0%0.0% 1.8% 0.0% 0.0% d6-elo 80.6% 86.6% 77.7% 79.6% 79.4% 77.5% d5-des93.7% 92.1% 91.7% 91.4% 91.5% 92.6% d5-elo 93.7% 92.1% 91.7% 91.4% 91.5%92.6% d4-des 100.0% 90.6% 94.8% 94.0% 95.3% 94.4% GA7_Col_#22GA7_Col_#14 GA7_FAD2_#25 GA7_FAD2_#21 GA7_FAD2_#18 d12-des 70.2% 72.7%45.9% 69.5% 53.7% d15-des 72.7% 77.2% 79.7% 66.0% 88.1% Omega-6 d6-des1.3% 1.0% 1.6% 1.1% 1.1% (d9-elo) 1.8% 1.7% 1.2% 2.7% 0.9% d6-elo 70.3%74.5% 85.5% 66.1% 88.0% d5-des 100.0% 100.0% 100.0% 100.0% 100.0% d5-elo100.0% 100.0% 100.0% 100.0% 100.0% d4-des 0.0% 0.0% 0.0% 0.0% 0.0%Omega-3 d6-des 24.7% 23.6% 27.1% 21.9% 21.0% (d9-elo) 2.0% 2.2% 0.0%2.6% 2.1% d6-elo 72.7% 73.0% 76.7% 77.4% 79.2% d5-des 89.6% 92.4% 88.0%91.8% 91.0% d5-elo 89.6% 92.4% 88.0% 91.8% 91.0% d4-des 95.8% 96.9%93.1% 92.9% 94.2%

TABLE 6 Compiled data from the total seed lipid profiles fromindependent transgenic seed shown in Table 2. Calculations do notinclude the ‘minor fatty acids’ in Table 4. Parameter pJP3404_Col_#1pJP3404_FAD2_#31 GA7_Col_#7 GA7_Col_#34 GA7_Col_#2 GA7_Col_#10 total w3(% of total FA) 21.8 18.8 34.9 35.6 32.9 39.1 total w6 (% of total FA)32.9 31.0 17.9 17.7 19.9 15.2 w3/w6 ratio 0.66 0.61 1.95 2.01 1.65 2.57w6/w3 ratio 1.51 1.65 0.51 0.50 0.60 0.39 total novel w3 (% of total FA)5.2 3.9 5.3 6.0 5.4 6.7 total novel w6 (% of total FA) 4.0 2.7 1.5 1.61.7 1.5 novel w3/w6 ratio 1.30 1.44 3.53 3.75 3.18 4.47 novel w6/w3ratio 0.77 0.69 0.28 0.27 0.31 0.22 OA to EPA efficiency 4.8% 3.5% 4.3%4.4% 4.7% 5.4% OA to DHA efficiency 4.5% 3.0% 3.7% 3.8% 4.1% 4.8% LA toEPA efficiency 6.9% 5.6% 6.6% 6.8% 7.2% 8.1% LA to DHA efficiency 6.6%4.8% 5.7% 5.8% 6.3% 7.2% ALA to EPA efficiency 17.4% 14.9% 10.0% 10.1%11.6% 11.3% ALA to DHA efficiency 16.5% 12.8% 8.6% 8.7% 10.0% 10.0%total saturates 14.1 14.0 14.4 14.1 14.4 13.5 total monounsaturates 29.334.7 31.4 31.2 31.4 31.0 total polyunsaturates 54.7 49.8 52.8 53.3 52.854.3 total C20 17.4 20 19.7 20.4 20.1 18.7 total C22 6.4 4.5 4.6 4.7 5.15.5 C20/C22 ratio 2.72 4.44 4.28 4.34 3.94 3.40 Parameter GA7_Col_#22GA7_Col_#14 GA7_FAD2_#25 GA7_FAD2_#21 GA7_FAD2_#18 total w3 (% of totalFA) 40.9 43.5 30.0 36.2 38.8 total w6 (% of total FA) 15.4 12.9 7.6 18.65.2 w3/w6 ratio 2.66 3.37 3.95 1.95 7.46 w6/w3 ratio 0.38 0.30 0.25 0.510.13 total novel w3 (% of total FA) 10.5 10.7 8.1 8.5 8.7 total novel w6(% of total FA) 1.7 1.5 1.0 2.0 0.9 novel w3/w6 ratio 6.18 7.13 8.104.25 9.67 novel w6/w3 ratio 0.16 0.14 0.12 0.24 0.10 OA to EPAefficiency 7.9% 8.8% 6.3% 6.4% 6.7% OA to DHA efficiency 6.8% 7.9% 5.2%5.5% 5.8% LA to EPA efficiency 11.4% 12.2% 13.8% 9.3% 12.7% LA to DHAefficiency 9.8% 11.0% 11.4% 8.0% 10.9% ALA to EPA efficiency 15.6% 15.9%17.3% 14.1% 14.4% ALA to DHA efficiency 13.4% 14.3% 14.3% 12.2% 12.4%total saturates 13.3 15.1 12.0 14.6 12.0 total monounsaturates 28.7 26.648.7 29.1 42.3 total polyunsaturates 56.3 56.4 37.6 54.8 44.0 total C2018.5 17.8 21.8 21 20.7 total C22 7.2 7.8 6.1 6.4 6.7 C20/C22 ratio 2.572.28 3.57 3.28 3.09

TABLE 7 Fatty acid composition of total seed lipid from independenttransgenic T₃ and T₄ Arabidopsis progeny seeds obtained from plant linesas in Table 3. The error shown in the T₄ generation denotes the SD of n= 10. GA7_Col_7.2 GA7_Col_34.2 GA7_Col_10.13 GA7_Col_22.2 GA7_Col_14.1916:0 9.8 9.0 9.5 11.2 10.4 18:0 4.0 3.8 4.2 3.4 3.5 18:1n7 2.0 1.9 2.22.9 2.5 20:0 2.2 1.9 1.7 1.4 2.3 20:1d13 1.4 1.3 1.2 1.6 2.5 20:1d9/1113.6 14.7 12.4 9.5 13.0 22:1d13 1.2 1.2 0.8 0.6 1.6 Other minor 1.8 1.51.5 2.1 2.6 18:1d9 5.5 6.7 6.8 4.6 6.9 18:2ω6 7.5 7.9 7.4 5.6 14.818:3ω3 33.7 33.7 36.1 31.5 26.1 18:3ω6 0.2 0.2 0.2 0.4 0.1 20:2ω6 1.01.0 0.7 0.7 1.4 20:3ω6 0 0 0 0 0 20:4ω6 0 0 0 0 0 22:4ω6 0 0 0 0 022:5ω6 0 0 0 0 0 18:4ω3 3.1 2.6 3.0 5.3 3.3 20:3ω3 1.4 1.3 1.2 1.3 1.220:4ω3 0.7 0.6 0.6 0.9 0.2 20:5ω3 0.9 0.9 0.7 1.9 0.8 22:5ω3 0.7 0.6 0.61.0 0.4 22:6ω3 9.5 9.2 9.4 13.9 6.6 GA7_FAD2- GA7_FAD2- GA7_FAD2- T₄Col_22.2 T₄ Col_22.2 25.10 21.2 18.14 (mean ± SD) best line 16:0 8.110.7 7.7 10.6 ± 0.9  12.2 18:0 3.5 3.8 3.3 3.5 ± 0.4 3.6 18:1n7 1.7 2.21.6 2.3 ± 0.2 2.6 20:0 1.8 2.0 1.9 1.9 ± 0.3 2.0 20:1d13 1.2 1.4 1.3 1.6± 0.2 1.9 20:1d9/11 15.7 12.4 18.4 11.7 ± 1.7  9.5 22:1d13 1.0 1.1 1.50.9 ± 0.1 0.8 Other minor 1.7 1.9 1.6 1.9 ± 0.1 2.3 18:1d9 11.3 4.2 11.54.6 ± 1.0 3.3 18:2ω6 5.8 8.9 5.6 5.3 ± 0.9 4.3 18:3ω3 28.3 28.9 30.831.0 ± 1.1  29.5 18:3ω6 0.3 0.6 0.1 0.4 ± 0.1 0.4 20:2ω6 0.6 1.2 0.6 0.9± 0.1 0.9 20:3ω6 0 0 0 20:4ω6 0 0 0 22:4ω6 0 0 0 0.1 ± 0.0 0.1 22:5ω6 00 0 18:4ω3 3.7 5.2 2.6 4.8 ± 0.9 5.5 20:3ω3 1.1 1.3 1.3 1.5 ± 0.2 1.720:4ω3 1.7 0.9 0.9 0.8 ± 0.2 0.8 20:5ω3 1.2 1.0 0.8 1.5 ± 0.3 1.8 22:5ω30.8 0.6 0.5 1.1 ± 0.2 1.5 22:6ω3 10.3 11.5 7.9 13.3 ± 1.6  15.1

Enzymatic conversion efficiencies for each enzyme step in the pathwayfor production of DHA from oleic acid are shown in Table 8 for the T₃seeds with the higher DHA levels. The Δ12-desaturase conversionefficiency in seeds of line 22.2 was 81.6% and the ω3-desaturaseefficiency was 89.1%, both of them remarkably high and indicating thatthese fungal (yeast) enzymes were able to function well in developingseeds. The activities of the other exogenous enzymes in the DHA pathwaywere similarly high for ω3 substrates with the Δ6-desaturase acting at42.2% efficiency, Δ6-elongase at 76.8%, Δ5-desaturase at 95.0%,Δ5-elongase at 88.7% and Δ4-desaturase at 93.3% efficiency. TheΔ6-desaturase activity on the (06 substrate LA was much lower, with theΔ6-desaturase acting at only 0.7% conversion efficiency on LA. GLA waspresent at a level of only 0.4% and was the only new ω6 product asidefrom 20:2ω6 detected in the T₃ seeds with the highest DHA content.Compiled data from the total seed lipid profiles from independenttransgenic seed (Table 7) are shown in Table 9. This data for the linewith the greatest DHA level included a total ω6 FA (including LA) tototal ω3 FA (including ALA) ratio of 0.10. The new ω6 FA (excluding LA)to new ω3 FA (excluding ALA) ratio in the lipid of this line was 0.05.Total polyunsaturated fatty acid levels were more than 50% in theselines, and greater than 60% in at least 4 of the lines. Overallconversion efficiencies were calculated to be: OA to EPA=21.8%, OA toDHA=18.0%, LA to EPA=26.9%, LA to DHA=22.2%, ALA to EPA=30.1%, ALA toDHA=24.9%.

TABLE 8 Conversion efficiencies of the individual enzymatic steps forthe production of DHA from oleic acid, observed in total seed lipid fromtransgenic T₃ Arabidopsis seeds as in Table 7. GA7_Col_7.2 GA7_Col_34.2GA7_Col_10.13 GA7_Col_22.2 GA7_Col_14.19 d12-des 75.4% 73.1% 75.7% 81.6%73.4% d15-des 85.3% 84.4% 86.2% 89.1% 70.2% Omega-6 d6-des 0.3% 0.3%0.3% 0.7% 0.3% (d9-elo) 1.7% 1.7% 1.2% 1.2% 2.6% d6-elo d5-des d5-elod4-des Omega-3 d6-des 30.7% 29.3% 28.2% 42.2% 30.2% (d9-elo) 2.7% 2.7%2.3% 2.4% 3.0% d6-elo 79.0% 81.1% 79.0% 76.8% 70.9% d5-des 94.0% 94.6%94.5% 95.0% 97.9% d5-elo 91.9% 91.7% 93.6% 88.7% 89.5% d4-des 93.2%93.7% 94.4% 93.3% 93.7% GA7_FAD2- GA7_FAD2- GA7_FAD2- T₄ Col_22.2 T₄Col_22.2 25.10 21.2 18.14 (mean) best line d12-des 66.6% 78.5% 63.1%67.6% 82.7% d15-des 87.5% 82.2% 87.6% 81.0% 90.9% Omega-6 d6-des 0.6%1.0% 0.2% 1.3% 0.7% (d9-elo) 1.1% 2.0% 1.3% 1.6% 1.5% d6-elo d5-desd5-elo d4-des Omega-3 d6-des 38.5% 40.0% 29.2% 41.0% 45.7% (d9-elo) 2.3%2.7% 2.9% 2.8% 3.1% d6-elo 79.2% 73.2% 79.1% 77.5% 77.7% d5-des 87.8%93.3% 91.1% 95.0% 95.8% d5-elo 89.9% 92.2% 91.6% 90.8% 90.2% d4-des92.5% 95.0% 93.9% 92.2% 90.9%

TABLE 9 Compiled data from the total seed lipid profiles fromindependent transgenic seed shown in Table 5. Calculations do notinclude the ‘minor fatty acids’ in Table 7. Parameter GA7-Col_7.2GA7-Col_34.2 GA7-Col_10.13 GA7-Col_22.2 GA7-Col_14.19 total w3 (% oftotal FA) 50.0 48.9 51.6 55.8 38.6 total w6 (% of total FA) 8.7 9.1 8.36.7 16.3 w3/w6 ratio 5.75 5.37 6.22 8.33 2.37 w6/w3 ratio 0.17 0.19 0.160.12 0.42 total novel w3 (% of total FA) 16.3 15.2 15.5 24.3 12.5 totalnovel w6 (% of total FA) 1.2 1.2 0.9 1.1 1.5 novel w3/w6 ratio 13.5812.67 17.22 22.09 8.33 novel w6/w3 ratio 0.07 0.08 0.06 0.05 0.12 OA toEPA efficiency 14.1% 13.3% 13.4% 21.8% 10.2% OA to DHA efficiency 12.0%11.4% 11.8% 18.0% 8.6% LA to EPA efficiency 18.9% 18.4% 17.9% 26.9%14.2% LA to DHA efficiency 16.2% 15.9% 15.7% 22.2% 12.0% ALA to EPAefficiency 22.2% 21.9% 20.7% 30.1% 20.2% ALA to DHA efficiency 19.0%18.8% 18.2% 24.9% 17.1% total saturates 16.0 14.7 15.4 16.0 16.2 totalmonounsaturates 23.7 25.8 23.4 19.2 26.5 total polyunsaturates 58.7 58.059.9 62.5 54.9 total C20 19 19.8 16.8 15.9 19.1 total C22 11.4 11 10.815.5 8.6 C20/C22 ratio 1.67 1.80 1.56 1.03 2.22 GA7-FAD2- GA7-FAD2-GA7-FAD2- T₄ Col_22.2 T₄ Col_22.2 Parameter 25.10 21.2 18.14 (mean ± SD)best line total w3 (% of total FA) 47.1 49.4 44.8 54.0 55.9 total w6 (%of total FA) 6.7 10.7 6.3 6.7 5.7 w3/w6 ratio 7.03 4.62 7.11 8.06 9.81w6/w3 ratio 0.14 0.22 0.14 0.12 0.10 total novel w3 (% of total FA) 18.820.5 14.0 23.0 26.4 total novel w6 (% of total FA) 0.9 1.8 0.7 1.4 1.4novel w3/w6 ratio 20.89 11.39 20.00 16.43 18.86 novel w6/w3 ratio 0.050.09 0.05 0.06 0.05 OA to EPA efficiency 15.0% 16.8% 11.2% 20.4% 24.5%OA to DHA efficiency 12.6% 14.8% 9.6% 17.1% 20.1% LA to EPA efficiency22.9% 21.8% 18.0% 26.2% 29.9% LA to DHA efficiency 19.1% 19.1% 15.5%21.9% 24.5% ALA to EPA efficiency 26.1% 26.5% 20.5% 29.4% 32.9% ALA toDHA efficiency 21.9% 23.3% 17.6% 24.6% 27.0% total saturates 13.4 16.512.9 16.0 17.8 total monounsaturates 30.9 21.3 34.3 21.1 18.1 totalpolyunsaturates 53.8 60.1 51.1 60.7 61.6 total C20 21.5 18.2 23.3 1816.6 total C22 12.1 13.2 9.9 15.4 17.5 C20/C22 ratio 1.78 1.38 2.35 1.170.95

T₃ seeds from the pJP3416-GA7 line 22.2 in the Columbia background,which were progeny from T₂ line 22, were sown directly to soil and thefatty acid composition of mature seed from the resultant T₃ plantsanalysed by GC. The average DHA level of these seeds was 13.3%±1.6(n=10) as a percentage of total fatty acids in the seed lipid. As shownin Table 6 (right hand column), the line with the highest level of DHAcontained 15.1% DHA in the total fatty acids of the seed lipid. Theenzymatic conversion efficiencies are shown in Table 8 for each step inthe production of DHA from oleic acid.

The total ω6 FA (including LA) to ω3 FA (including ALA) ratio in theline with the highest DHA level was 0.102. The new ω6 FA (excluding LA)to new ω3 FA (excluding ALA) ratio in the line with the highest DHAlevel was 0.053. The level of total saturated fatty acids was about17.8% and the level of monounsaturated fatty acids was about 18.1%. Thelevel of total ω6-fatty acids was about 5.7% and the level of ω3-fattyacids was about 55.9%. Overall conversion efficiencies were calculatedto be: OA to EPA=24.5%, OA to DHA=20.1%, LA to EPA=29.9%, LA toDHA=24.5%, ALA to EPA=32.9%, ALA to DHA=27.0%. Total omega-3 fatty acidswere found to accumulate to 55.9% of total fatty acids whereas omega-6fatty acids were 5.7% of the total profile.

Southern blot hybridisation analysis was performed. The results showedthat the high-accumulating DHA lines were either single- or double-copyfor the T-DNA from the pJP3416-GA7 construct with the exception oftransgenic line Columbia#22, which had three T-DNA insertions in thegenome of the Arabidopsis plant. The T₅ generation seed was alsoanalysed and found to have up to 13.6% DHA in the total seed lipids. TheGA7 construct was found to be stable across multiple generations interms of DHA production capability.

Determination of Oil Content in Transgenic A. thaliana DHA Lines

The oil content of transgenic A. thaliana seeds with various levels ofDHA was determined by GC as described in Example 1. The data are shownin FIG. 6, graphing the oil content (% oil by weight of seed) againstthe DHA content (as a percentage of total fatty acids). Up to 26.5 mg ofDHA per gram of seed was observed (Table 10). The oil content of thetransgenic Arabidopsis seeds was found to be negatively correlated withDHA content. The amount of DHA per weight of seed was greater in thetransformed seeds with a DHA level of about 9% relative to the seedswith about 14% DHA. Whether this would be true for seeds other thanArabidopsis has not been determined.

TABLE 10 Proportion and amount of DHA in GA7- transformed Arabidopsisseeds. Oil content DHA content DHA content (% oil per per weight (% ofTFA) g seeds) (mg/g seed) GA7/col 22.2-1 14.2 14.89 20.2 GA7/col 22.2-214.3 15.02 20.5 GA7/col 22.2-3 14.0 15.92 21.2 GA7/col 10.15-1 8.7 30.2325.06 GA7/col 10.15-2 8.6 31.25 25.77 GA7/col 10.15-3 8.8 31.70 26.49

Example 3. Stable Expression of a Transgenic DHA Pathway in Camelinasativa Seeds

The binary vector pJP3416-GA7 as described above was introduced into A.tumefaciens strain AGL1 and cells from a culture of the transformedAgrobacterium used to treat C. sativa flowering plants using a floraldip method for transformation (Lu and Kang, 2008). After growth andmaturation of the plants, the T₁ seeds from the treated plants wereharvested, sown onto soil and the resultant plants treated by sprayingwith the herbicide BASTA to select for plants which were transgenic for,and expressing, the bar selectable marker gene present on the T-DNA ofpJP3416-GA7. Surviving T₁ plants which were tolerant to the herbicidewere grown to maturity after allowing them to self-fertilise, and theresultant T₂ seed harvested. Five transgenic plants were obtained, onlythree of which contained the entire T-DNA.

Lipid was extracted from a pool of approximately twenty seeds from eachof the three plants that contained the entire T-DNA. Two of the pooledsamples contained very low, barely detectable levels of DHA, but thethird pool contained about 4.7% DHA (Table 12). Therefore, lipid wasextracted from 10 individual T₂ seeds from this plant and the fatty acidcomposition analysed by GC. The fatty acid composition data of theindividual seeds for this transformed line is also shown in Table 11.Compiled data from the total seed lipid profiles (Table 11) are shown inTable 12.

TABLE 11 Fatty acid composition of total seed lipids from transgenic T₂Camelina sativa seeds transformed with the T-DNA from pJP3416-GA7. Thefatty acid composition is shown for a pooled seed batch (FD5.46) and for10 single seeds ranked (left to right) from highest to lowest DHA.FD5.46 Fatty acid pooled # 2 # 4 # 8 # 7 # 9 # 1 # 3 # 5 # 6 # 10 14:0 00.2 0.2 0.1 0.2 0.2 0.2 0.2 0.1 0.2 0.2 16:0 11.6 12.1 12.3 12.1 13.212.3 12.8 11.9 11.4 11.5 11.7 16:1 0.2 0.0 0.1 0.1 0.0 0.2 0.0 0.2 0.20.2 0.2 16:3 0.3 0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 18:0 3.7 3.3 3.23.2 3.0 3.1 3.2 3.3 3.1 3.2 3.2 18:1 10.8 8.0 8.0 8.6 8.5 9.4 11.0 10.28.3 9.4 8.6 18:1d11 1.7 1.3 1.4 1.4 1.7 1.4 1.5 1.3 1.3 1.3 1.3 18:224.7 18.2 19.5 19.2 18.5 20.1 23.8 32.2 30.3 29.8 31.6 18:3ω3 27.4 26.726.6 27.3 28.9 28.2 27.4 28.3 29.2 29.5 28.2 18:3ω6 0.2 1.4 0.3 0.3 0.40.2 0.5 0.0 0.5 0.4 0.6 20:0 1.6 1.4 1.3 1.4 1.2 1.4 1.4 1.8 2.1 1.9 2.018:4ω3 2.2 6.8 6.4 5.7 7.2 5.7 4.1 0.0 0.0 0.0 0.0 20:1d11 5.3 4.4 4.64.8 3.3 4.1 3.5 4.4 6.1 5.8 5.5 20:1iso 0.4 0.3 0.3 0.3 0.3 0.3 0.0 0.50.6 0.5 0.5 20:2ω6 0.8 0.8 0.9 0.8 0.6 0.8 0.7 1.3 1.5 1.4 1.4 20:3ω30.6 0.8 0.8 0.8 0.7 0.8 0.7 0.6 0.7 0.7 0.6 22:0 0.4 0.5 0.5 0.5 0.4 0.50.5 0.6 0.6 0.6 0.6 20:4ω3 0.2 0.3 0.3 0.3 0.4 0.4 0.5 0.0 0.0 0.0 0.022:1 1.1 1.1 1.2 1.1 0.5 0.9 0.8 1.6 2.2 1.9 2.0 20:5ω3 0.7 1.3 1.6 1.51.6 1.1 1.7 0.0 0.0 0.0 0.1 22:2ω6 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.2 0.30.2 0.2 22:4ω6 + 0.3 0.2 0.3 0.3 0.0 0.3 0.0 0.4 0.6 0.5 0.5 22:3ω3 24:00.3 0.3 0.3 0.3 0.0 0.3 0.0 0.4 0.4 0.4 0.4 24:1 0.3 0.4 0.4 0.3 0.0 0.30.0 0.5 0.6 0.5 0.5 22:5ω3 0.3 1.1 1.2 1.1 1.1 0.9 0.8 0.0 0.0 0.0 0.022:6ω3 4.7 9.0 8.5 8.3 8.3 7.1 4.9 0.0 0.0 0.0 0.0

TABLE 12 Compiled data from the total seed lipid profiles fromtransgenic seed shown in Table 11. Calculations do not include the‘minor fatty acids’ in Table 11. FD5.46 Parameter pooled # 2 # 4 # 8 # 7# 9 # 1 # 3 # 5 # 6 # 10 total w3 (% of total FA) 36.1 46 45.4 45 48.244.2 40.1 28.9 29.9 30.2 28.9 total w6 (% of total FA) 25.8 20.4 20.720.3 19.5 21.1 25 33.7 32.6 31.8 33.8 w3/w6 ratio 1.40 2.25 2.19 2.222.47 2.09 1.60 0.86 0.92 0.95 0.86 w6/w3 ratio 0.71 0.44 0.46 0.45 0.400.48 0.62 1.17 1.09 1.05 1.17 total novel w3 (% of total FA) 8.1 18.5 1816.9 18.6 15.2 12 0 0 0 0.1 total novel w6 (% of total FA) 1.1 2.2 1.21.1 1 1 1.2 1.5 2.3 2 2.2 novel w3/w6 ratio 7.36 8.41 15.00 15.36 18.6015.20 10.00 0.05 novel w6/w3 ratio 0.14 0.12 0.07 0.07 0.05 0.07 0.1022.00 OA to EPA efficiency 8.2% 15.6% 15.5% 15.1% 15.1% 12.8% 10.5% 0.0%0.0% 0.0% 0.1% OA to DHA efficiency 6.7% 12.3% 11.6% 11.5% 11.4% 10.0%7.0% 0.0% 0.0% 0.0% 0.0% LA to EPA efficiency 9.2% 17.2% 17.1% 16.7%16.2% 13.9% 11.4% 0.0% 0.0% 0.0% 0.2% LA to DHA efficiency 7.6% 13.6%12.9% 12.7% 12.3% 10.9% 7.5% 0.0% 0.0% 0.0% 0.0% ALA to EPA efficiency15.8% 24.8% 24.9% 24.2% 22.8% 20.6% 18.5% 0.0% 0.0% 0.0% 0.3% ALA to DHAefficiency 13.0% 19.6% 18.7% 18.4% 17.2% 16.1% 12.2% 0.0% 0.0% 0.0% 0.0%total saturates 17.6 17.8 17.8 17.6 18 17.8 18.1 18.2 17.7 17.8 18.1total monounsaturates 19.8 15.5 16 16.6 14.3 16.6 16.8 18.7 19.3 19.618.6 total polyunsaturates 62.5 66.6 66.4 65.6 67.7 65.6 65.1 63 63.162.5 63.2 total C20 9.6 9.3 9.8 9.9 8.1 8.9 8.5 8.6 11 10.3 10.1 totalC22 5.4 10.3 10 9.7 9.4 8.3 5.7 0.6 0.9 0.7 0.7 C20/C22 ratio 1.78 0.900.98 1.02 0.86 1.07 1.49 14.33 12.22 14.71 14.43

DHA was present in six of the 10 individual seeds. The four other seedsdid not have DHA and were presumed to be null segregants which did nothave the T-DNA, based on hemizygosity of the T-DNA insertion in theparental plant. Extracted lipid from the single seed with the highestlevel of DHA had 9.0% DHA while the sum of the percentages for EPA, DPAand DHA was 11.4%. The sum of the percentages for the new ω3 fatty acidsproduced in this seed as a result of the transformation (SDA, ETrA, ETA,EPA, DPA, DHA) was 19.3% whilst the corresponding sum for the new ω6fatty acids (GLA, EDA, DGLA, ARA and any ω6 elongation products) was2.2%—only GLA and EDA were detected as new ω6 fatty acids. The total ω6FA (including LA) to 03 FA (including ALA) ratio was found to be 0.44.The new ω6 FA (excluding LA) to new ω3 FA (excluding ALA) ratio in theseed with the highest DHA level was 0.12. The level of total saturatedfatty acids was about 17.8% and the level of monounsaturated fatty acidswas about 15.5%. The level of total ω6-fatty acids was about 20.4% andthe level of ω3-fatty acids was about 46%. Overall conversionefficiencies were calculated to be: OA to EPA=15.6%, OA to DHA=12.3%, LAto EPA=17.2%, LA to DHA=13.6%, ALA to EPA=24.8%, ALA to DHA=19.6%.

Homozygous seed from this line was obtained in the T₄ generation. Up to10.3% DHA was produced in event FD5-46-18-110 with an average of 7.3%DHA observed across the entire T₄ generation.

Homozygous seed was planted out across several glasshouses to generate atotal of over 600 individual plants. Oil is being extracted from theseed using a variety of methods including soxhlet, acetone and hexaneextractions.

Since the number of independently transformed lines of C. sativaobtained as described above was low, further experiments to transform C.sativa with pJP3416-GA7 are performed. The inventors predict that DHAlevels of greater than 10% as a percentage of total fatty acids in seedoil will be achieved in further transformed lines, and plants which arehomozygous for the T-DNA to 20% DHA. Twenty C. sativa GA7_modH eventswere generated and seed is being analysed for DHA content. ThreeGA7_modB events were generated and analysis of the T1 seed from eventCMD17.1 revealed a pooled seed DHA content of 9.8%. The highest singleseed DHA value was found to be 13.5%.

Example 4. Stable Expression of Transgenic DHA Pathways in Brassicanapus Seeds

B. napus Transformation and Analysis of Fatty Acid Composition UsingSingle Vector

The binary vector pJP3416-GA7 was used to generate transformed Brassicanapus plants and seeds from the plants. The vector pJP3416-GA7 asdescribed above was introduced into Agrobacterium tumefaciens strainAGL1 via standard electroporation procedures. Cultures of the transgenicAgrobacterium cells were grown overnight at 28° C. in LB medium withagitation at 150 rpm. The bacterial cells were collected bycentrifugation at 4000 rpm for 5 minutes, washed with Winans AB medium(Winans, 1988) and re-suspended in 10 mL of Winans AB medium (pH 5.2)and growth continued overnight in the presence of kanamycin (50 mg/L),rifampicin (25 mg/L) and 100 μM acetosyringone. Two hours beforeinfection of the Brassica cells, spermidine (120 mg/L) was added and thefinal density of the bacteria adjusted to an OD 600 nm of 0.3-0.4 withfresh AB media. Freshly isolated cotyledonary petioles from 8-day oldBrassica napus seedlings grown on ½ MS (Murashige and Skoog, 1962) orhypocotyl segments preconditioned by 3-4 days on MS media with 1 mg/Lthidiazuron (TDZ) and 0.1 mg/L α-naphthaleneacetic acid (NAA) wereinfected with 10 mL Agrobacterium cultures for 5 minutes. The explantsinfected with Agrobacterium were then blotted on sterile filter paper toremove the excess Agrobacterium and transferred to co-cultivation media(MS media with 1 mg/L TDZ, 0.1 mg/L NAA and 100 μM acetosyringone)supplemented with or without different antioxidants (L-cysteine 50 mg/Land ascorbic 15 mg/L). All the plates were sealed with parafilm andincubated in the dark at 23-24° C. for 48 hrs.

The treated explants were then washed with sterile distilled watercontaining 500 mg/L cefotaxime and 50 mg/L timentin for 10 minutes,rinsed in sterile distilled water for 10 minutes, blotted dry on sterilefilter paper, transferred to pre-selection media (MS containing 1 mg/LTDZ, 0.1 mg/L NAA, 20 mg/L adenine sulphate (ADS), 1.5 mg/L AgNO₃, 250mg/L cefotaxime and 50 mg/L timentin) and cultured for five days at 24°C. with a 16 h/8 h photoperiod. They were then transferred to selectionmedia (MS containing 1 mg/L TDZ, 0.1 mg/L NAA, 20 mg/L ADS, 1.5 mg/LAgNO₃, 250 mg/L cefotaxime and 50 mg/L timentin) with 1.5 mg/Lglufosinate ammonium as the agent for selection of transformed cells,and cultured for 4 weeks at 24° C. with 16 h/8 h photoperiod with abiweekly subculture on to the same media. Explants with green calluswere transferred to shoot initiation media (MS containing 1 mg/Lkinetin, 20 mg/L ADS, 1.5 mg/L AgNO₃, 250 mg/L cefotaxime, 50 mg/Ltimentin and 1.5 mg/L glufosinate ammonium) and cultured for another 2-3weeks. Shoots emerging from the resistant explants were transferred toshoot elongation media (MS media with 0.1 mg/L gibberelic acid, 20 mg/LADS, 1.5 mg/L AgNO₃, 250 mg/L cefotaxime and 1.5 mg/L glufosinateammonium) and cultured for another two weeks. Healthy shoots 2-3 cm longwere selected and transferred to rooting media (½ MS containing 1 mg/LNAA, 20 mg/L ADS, 1.5 mg/L AgNO₃ and 250 mg/L cefotaxime) and culturedfor 2-3 weeks. Well established shoots with roots were transferred topots containing seedling raising mix and grown in a growth cabinet fortwo weeks and subsequently transferred to a glasshouse. Approximately 40(To) plants transformed with the GA7 construct were obtained by thismethod.

Plants were grown to maturity after being allowed to self-fertilise.Seeds obtained from transformed plants were analysed for fatty acidcomposition in their seedoil as described in Example 1. Data for atransformed line with the highest DHA level are shown in Table 13. DHAlevels on average were significantly lower in the seedoil of the B.napus seeds transformed with the T-DNA from pJP3416-GA7 than in A.thaliana seeds (Example 2) or Camelina seeds (Example 3) transformedwith the same construct. The highest level of DHA in approximately 40lines was found to be 1.52% with the majority of the transgenic lineshaving detectable DHA. It was noted that there was a substantialaccumulation of ALA, about 35% of the total fatty acids, in these seedswhich was not being converted efficiently to SDA or following productsin the pathway.

Fatty acid profile analysis of single B. napus seeds from a T₁ event,CT125-2, was performed to better determine the amount of DHA produced intransgenic seeds. Seeds were found to contain between 0% (null seeds)and 8.5% DHA (Table 13).

Some of the seeds from the plant line CT116 as well as other transgeniclines showing DHA production were sown to produce progeny plants. RT-PCRwas performed on total RNA isolated from developing embryos from theseplants in order to determine why the GA7 construct performed poorly forDHA production relative to transgenic A. thaliana and C. sativa havingthe same construct, and poorly relative to the combination of the geneson pJP3115 and pJP3116 (below). RT-PCR was performed on total RNA usinga one-step RT-PCR kit (Invitrogen) and gene-specific primers targetingeach transgene. This confirmed that each of the genes in the GA7construct was expressed well in the B. napus transformants except forthe Δ6-desaturase which was poorly expressed in the majority oftransformed seeds. The other genes from this construct functioned wellin both B. napus and A. thaliana seeds, for example the Δ12- andΔ15-desaturases which functioned to produce increased levels of LA andALA in the seeds whilst decreasing oleic acid levels. A representativeRT-PCR gel is shown in FIG. 7 which clearly shows the low expression ofthe Δ6-desaturase relative to the other transgenes from pJP3416-GA7.

Transgenic plants and seed which are homozygous for the transgenes aregenerated by planting out progeny from the lines with the highest DHA.

TABLE 13 Fatty acid composition as a percentage of total fatty acids inseedoil from independent T₁ Brassica napus seed transformed withpJP3416-GA7, lines CT116-11 and CT-125-2 compared to wild- type(untransformed) control. 22:6ω3 is DHA. Data from single CT125-2 B.napus seeds is denoted by ‘SS’. CT116- CT125- CT125-2 CT125-2 CT125-2Control 11 2 #2 SS #3 SS #10 SS 14:0 0.1 0.2 0.1 0.1 0.1 0.1 16:0 4.37.2 5.2 6.5 4.7 7.7 16:1 0.2 0.5 0.4 0.3 0.3 0.8 16:3 0.2 0.2 0.2 0.10.2 0.2 18:0 2.1 2.2 2.4 2.3 2.3 2.8 18:1d9 59.1 27.0 38.1 34.0 19.314.8 18:1d11 3.7 6.6 4.2 4.4 4.3 9.6 18:2 19.7 14.1 16.6 13.9 10.2 10.218:3ω3 8.3 35.2 27.7 34.1 49.5 37.9 20:0 0.6 0.5 0.6 0.4 0.3 0.7 18:4ω30.0 0.9 0.3 0.5 0.6 2.6 20:1d11 1.2 1.1 1.0 1.0 0.8 0.6 20:1is0 0.2 0.10.2 20:2ω6 0.1 0.1 0.1 0.1 0.1 0.1 20:3ω3 1.3 0.7 0.8 1.6 0.9 22:0 0.30.4 0.3 0.1 0.1 0.4 20:4ω3 0.1 0.3 0.4 0.6 0.5 22:1 20:5ω3 0.1 0.322:3ω3 0.1 24:0 0.2 0.4 0.3 0.1 0.1 0.3 24:1 0.1 0.3 0.1 0.1 0.2 0.122:5ω3 0.1 0.1 0.1 0.1 0.5 22:6ω3 1.52 1.2 1.3 2.7 8.5B. napus Transformation and Analysis of Fatty Acid Composition Using TwoVectors

In another experiment in B. napus and as an alternative format forintroducing the transgenes, the binary vectors pJP3115 and pJP3116 asdescribed in WO 2010/057246 were used to separately generate transformedB. napus plants and transformed seeds were obtained from the plants. TheT-DNA on pJP3115 comprised chimeric genes encoding the Crepis palestinaΔ12-desaturase, Micromonas pusilla Δ6-desaturase, Pyramimonas cordataΔ6-elongase and Pavlova salina Δ5-desaturase and the T-DNA on pJP3116contained chimeric genes encoding Perilla frutescens Δ15-desaturase,Pyramimonas cordata Δ5-elongase and Pavlova salina Δ4-desaturase. Thetwo T-DNAs, when present together and expressed in developing seeds,formed a 7-gene pathway for producing DHA from endogenously producedoleic acid. These vectors were introduced into Agrobacterium tumefaciensstrain AGL1 via standard electroporation procedures and the transformedcells used independently to transform B. napus using the method asdescribed above to generate stably transformed T₀ plants. 29 pJP3115 and19 pJP3116 transformants were obtained and these plants were grown tomaturity and seeds obtained after self-fertilisation were analysed forfatty acid composition in their seedoil. Transformation with the T-DNAfrom pJP3115 was expected to result in EPA production from endogenouslyproduced ALA whilst transformation with the T-DNA from pJP3116 wasexpected to result in increased ALA production from LA. Several plantswere identified which displayed these phenotypes. The majority of eventsdisplayed a decreased OA/increased LA phenotype due to Δ12 desaturationwith a low level of EPA production. Up to 2.6% EPA was observed inpJP31115 transgenic pooled seed. Similarly, the majority of pJP3116events were found to have an elevated ALA phenotype due toΔ15-desaturase activity. Up to 18.5% ALA was found in pooled seedtransformed with the T-DNA from pJP3116.

T₁ plants from the lines with the highest levels of EPA and ALA werecrossed and the progeny seed (F1) from 24 recovered events analysed forDHA content. DHA was found in 17 of these events with up to 1.9% DHAfound in pooled seed from these events. Single-seed analysis wasperformed to determine the range of DHA production—the data are shown inTable 14. A large range of DHA levels were observed in the crossedprogeny, probably due to the hemizygous nature of the T-DNAs in theparental plants, so that some seeds did not receive both T-DNAs. Up to6.7% DHA was observed in total seed lipid.

TABLE 14 Fatty acid composition as a percentage of total fatty acids inseedoil from B. napus F1 single seeds that were from a cross of plantstransgenic for the T-DNA from pJP3115 with plants transgenic for theT-DNA from pJP3116. B1, B2 and B4 designate events. 0.0 = not detectableby the GC method. B1.1 B1.2 B1.3 B1.4-g B1.5-g B2.1 B2.2 B2.3g B2.4gB2.5g B3.1 B3.2 14:0 0.1 0.1 0.1 0.2 0.2 0.1 0.1 0.2 0.2 0.1 0.1 0.116:0 6.6 6.4 4.5 12.3 7.9 5.1 5.0 10.1 8.5 6.8 5.3 7.2 16:1 0.4 0.5 0.21.0 0.6 0.4 0.4 0.6 1.1 0.5 0.5 0.6 16:3 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.10.2 0.1 0.1 0.2 18:0 2.3 2.6 2.2 1.6 2.9 2.9 3.4 2.2 1.8 2.9 3.4 2.418:1 34.1 39.3 46.9 14.9 20.7 41.6 46.3 14.4 23.4 38.3 43.6 32.0 18:1d114.6 5.8 2.7 6.8 6.2 3.8 4.9 5.9 8.7 4.5 5.5 5.1 18:2 33.6 30.7 30.4 29.234.4 31.7 27.7 33.2 23.9 33.3 27.9 33.4 18:3ω6 0.2 0.3 0.1 0.4 0.4 0.20.2 0.7 0.1 0.2 0.2 0.3 18:3ω3 10.3 7.1 7.7 18.7 14.9 8.2 5.9 14.8 28.16.3 7.3 10.0 20:0 0.6 0.7 0.6 0.5 0.7 0.8 0.9 0.6 0.4 0.7 0.9 0.7 18:4ω30.2 0.1 0.1 0.8 0.5 0.2 0.2 0.8 0.0 0.2 0.2 0.2 20:1d11 1.0 1.1 1.1 0.70.8 1.1 1.1 0.5 0.9 1.1 1.1 0.9 20:1iso 0.1 0.1 0.0 0.1 0.1 0.1 0.1 0.10.3 0.1 0.1 0.1 20:2ω6 0.4 0.3 0.2 0.5 0.5 0.4 0.3 0.4 0.5 0.5 0.3 0.520:3ω6 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 20:4ω6 0.0 0.00.0 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.0 0.0 20:3ω3 1.8 1.6 1.1 2.8 2.1 1.11.0 2.7 0.7 1.4 0.9 1.6 22:0 0.3 0.4 0.3 0.3 0.4 0.4 0.5 0.3 0.3 0.4 0.50.4 20:4ω3 0.3 0.2 0.2 0.4 0.4 0.1 0.1 0.5 0.0 0.2 0.1 0.2 22:1 0.0 0.00.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 20:5ω3 0.0 0.0 0.0 0.1 0.1 0.00.0 0.2 0.0 0.0 0.0 0.0 22:2ω6 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.00.0 0.0 22:4ω6 0.1 0.2 0.1 0.2 0.2 0.1 0.1 0.4 0.2 0.2 0.1 0.2 24:0 0.30.4 0.2 0.2 0.3 0.3 0.3 0.3 0.4 0.4 0.4 0.3 22:5ω6 0.1 0.2 0.1 0.2 0.30.1 0.1 0.5 0.0 0.2 0.1 0.2 24:1 0.2 0.2 0.2 0.3 0.3 0.2 0.2 0.3 0.3 0.20.2 0.2 22:5ω3 0.7 0.7 0.3 2.1 1.6 0.3 0.4 3.2 0.0 0.5 0.4 1.2 22:6ω31.4 1.0 0.5 5.5 3.9 0.8 0.7 6.7 0.0 1.1 0.8 2.0

TABLE 15 Compiled data from the total seed lipid profiles fromtransgenic seed shown in Table 14. Calculations do not include the‘minor fatty acids’ in Table 14. Parameter B1.1 B1.2 B1.3 B1.4-g B1.5-gB2.1 B2.2 B2.3g B2.4g B2.5g B3.1 total w3 (% of total FA) 4.6 3.9 2.312.1 9 2.7 2.6 14.8 0.8 3.6 2.6 total w6 (% of total FA) 44.5 38.5 38.548.8 50.3 40.5 34.1 49.4 52.7 40.5 35.7 w3/w6 ratio 0.10 0.10 0.06 0.250.18 0.07 0.08 0.30 0.02 0.09 0.07 w6/w3 ratio 9.67 9.87 16.74 4.03 5.5915.00 13.12 3.34 65.88 11.25 13.73 total novel w3 (% of total FA) 2.6 21.1 8.9 6.5 1.4 1.4 11.4 0 2 1.5 total novel w6 (% of total FA) 10.5 7.57.9 19.1 15.4 8.4 6.1 15.8 28.3 6.7 7.5 novel w3/w6 ratio 0.25 0.27 0.140.47 0.42 0.17 0.23 0.72 0.00 0.30 0.20 novel w6/w3 ratio 4.04 3.75 7.182.15 2.37 6.00 4.36 1.39 3.35 5.00 OA to EPA efficiency 2.5% 2.1% 0.9%10.1% 6.9% 1.3% 1.3% 12.8% 1.9% 1.4% OA to DHA efficiency 1.7% 1.2% 0.6%7.2% 4.8% 0.9% 0.8% 8.5% 1.3% 1.0% LA to EPA efficiency 4.3% 4.0% 2.0%12.6% 9.4% 2.5% 3.0% 15.7% 3.6% 3.1% LA to DHA efficiency 2.9% 2.4% 1.2%9.0% 6.6% 1.9% 1.9% 10.4% 2.5% 2.1% ALA to EPA efficiency 47.7% 44.7%36.4% 68.1% 65.9% 44.0% 45.8% 72.1% 47.1% 50.0% ALA to DHA efficiency31.8% 26.3% 22.7% 48.7% 45.9% 32.0% 29.2% 47.9% 32.4% 33.3% totalsaturates 10.2 10.6 7.9 15.1 12.4 9.6 10.2 13.7 11.6 11.3 10.6 totalmonounsaturates 40.4 47 51.1 23.8 28.7 47.2 53 21.8 34.7 44.7 51 totalpolyunsaturates 49.2 42.5 40.9 61 59.4 43.3 36.8 64.3 53.7 44.2 38.4total C20 4.2 4 3.2 5.1 4.7 3.6 3.5 5.1 2.8 4 3.4 total C22 2.6 2.5 1.38.3 6.4 1.7 1.8 11.1 0.5 2.4 1.9 C20/C22 ratio 1.62 1.60 2.46 0.61 0.732.12 1.94 0.46 5.60 1.67 1.79

Compiled data from the total lipid profiles (Table 14) are shown inTable 15. From the data in Table 15, the total ω6 FA (including LA) toω3 FA (including ALA) ratio in the seed with the highest level of DHAwas 3.34. The new ω6 FA (excluding LA) to new ω3 FA (excluding ALA)ratio was 1.39. The level of total saturated fatty acids was about 13.7%and the level of monounsaturated fatty acids was about 21.8%. The levelof total ω6-fatty acids was about 46.4% and the level of ω3-fatty acidswas about 14.8%. Overall conversion efficiencies were calculated to be:OA to EPA=12.8%, OA to DHA=8.5%, LA to EPA=15.7%, LA to DHA=10.4%, ALAto EPA=72.1%, ALA to DHA=47.9%. The reduced efficiency of the ω6 fattyacids to ω3 fatty acids conversion observed in this experiment with thecombination of the pJP3115 and pJP3116 was thought to be due to a lowerefficiency of the plant Δ15-desaturase compared to the fungal Δ15/ω3desaturase (Examples 2 and 3) when combined with the genes forconversion of ALA to DHA.

Progeny from DHA-containing lines which are homozygous for all of theintroduced transgenes are generated for analysis.

Example 5. Modifications to T-DNAs Encoding DHA Pathways in Plant Seeds

In order to improve the DHA production level in B. napus beyond thelevels described in Example 4, the binary vectors pJP3416-GA7-modA,pJP3416-GA7-modB, pJP3416-GA7-modC, pJP3416-GA7-modD, pJP3416-GA7-modEand pJP3416-GA7-modF were constructed as follows. These binary vectorswere variants of the pJP3416-GA7 construct described in Example 2 andwere designed to further increase the synthesis of DHA in plant seeds,particularly by improving Δ6-desaturase and Δ6-elongase functions. SDAhad been observed to accumulate in some seed transformed with the GA7construct due to a relatively low elongation efficiency compared to theΔ5-elongase, so amongst other modifications, the two elongase genepositions were switched in the T-DNA.

The two elongase coding sequences in pJP3416-GA7 were switched in theirpositions on the T-DNA to yield pJP3416-GA7-modA by first cloning a newP. cordata Δ6-elongase cassette between the SbfI sites of pJP3416-GA7 toreplace the P. cordata Δ5-elongase cassette. This construct was furthermodified by exchanging the FP1 promoter driving the M. pusillaΔ6-desaturase with a conlinin Cnl2 promoter (pLuCnl2) to yieldpJP3416-GA7-modB. This modification was made in an attempt to increasethe Δ6-desaturase expression and thereby enzyme efficiency. It wasthought that the Cnl2 promoter might yield higher expression of thetransgene in B. napus than the truncated napin promoter.pJP3416-GA7-modC was produced by adding a second M. pusillaΔ6-desaturase cassette with slightly different codon usage (SEQ ID NO:15) and driven by the FP1 promoter, which was inserted at the PmeI sitejust inside the right border of pJP3416-GA7-modB. The secondΔ6-desaturase cassette was added to both pJP3416-GA7-modB andpJP3416-GA7-modF in order to increase the Δ6-desaturase expression leveland extend the time period during seed development for expression ofΔ6-desaturase by the use of multiple promoters. Different codon usageswere used in the two nucleotide sequences to result in the translationof the same protein sequence without risking co-suppression from similarcoding regions within the same T-DNA. pJP3416-GA7-modD andpJP3416-GA7-modE were similar variants in which a third MAR sequence,corresponding to nucleotides 16649-17816 of SEQ ID NO: 1, was added topJP3416-GA7 and pJP3416-GA7-modB, respectively, at the PmeI site.pJP3416-GA7-modF was produced by adding a second M. pusillaΔ6-desaturase cassette containing the native Δ6-desaturase nucleotidesequence and driven by the FP1 promoter at the PmeI site at the rightborder of pJP3416-GA7-modB. pJP3416-GA7-modG was made by first replacingthe M. pusilla Δ6-desaturase cassette with a Cnl2:P. cordata Δ5-elongasecassette by restriction cloning at the AscI-PacI sites. pJP3416-GA7-modGwas then made by replacing the original FAE1:P. cordata Δ5-elongasecassette with a FAE1:M. pusilla Δ6-desaturase cassette by restrictioncloning at the SbfI sites. The nucleotide sequences of the T-DNAs fromeach of these genetic constructs are shown as: pJP3416-GA7-modB (SEQ IDNO:2), pJP3416-GA7-modC (SEQ ID NO:3), pJP3416-GA7-modD (SEQ ID NO:4),pJP3416-GA7-modE (SEQ ID NO:5), pJP3416-GA7-modF (SEQ ID NO:6) andpJP3416-GA7-modG (SEQ ID NO:7).

The binary vectors pJP3416-GA7-modB, pJP3416-GA7-modC, pJP3416-GA7-modD,pJP3416-GA7-modE, pJP3416-GA7-modF and pJP3416-GA7-modG are used togenerate transformed Brassica somatic embryos and Brassica napus,Camelina sativa and Arabidopsis thaliana plants and progeny seeds. Datafor pJP3416-GA7-modB are shown in the next Example.

Eight transgenic pJP3416-GA7-modB A. thaliana events and 15 transgenicpJP3416-GA7-modG A. thaliana events were generated. Between 3.4% and7.2% DHA in pooled pJP3416-GA7-modB seed was observed and between 0.6and 4.1% DHA in pooled T₂ pJP3416-GA7-modG seed was observed. Several ofthe highest pJP3416-GA7-modB events were sown out on selectable mediaand surviving seedlings taken to the next generation. Seed is beinganalysed for DHA content. Since the pooled T1 seeds representedpopulations that were segregating for the transgenes and included anynull segregants, it is expected that the homozygous seeds from progenyplants will have increased levels of DHA, up to 20% of the total fattyacid content in the seed oil. The other modified constructs were used totransform A. thaliana. Although only a small number of transformed lineswere obtained, none yielded higher levels of DHA than the modBconstruct.

The pJP3416-GA7-modB construct was also used to generate transformed B.napus plants of cultivar Oscar and in a breeding line designated NX005.Ten independent transformed plants (T0) were obtained so far for theOscar transformation, and 20 independent lines for NX005. Seed (T1 seed)was harvested from these transgenic lines. Pools of seed were tested forlevels of DHA in the seed oil, and two lines which showed the highestlevels were selected, these were designated lines CT132.5 (in cultivarOscar) and CT133.15 (in NX005). Twenty seeds from CT132.5 and 11 seedsfrom CT133.15 were imbibed and, after two days, oil was extracted from ahalf cotyledon from each of the individual seeds. The other halfcotyledons with embryonic axes were kept and cultured on media tomaintain the specific progeny lines. The fatty acid composition in theoil was determined; the data is shown in Table 16 for CT132.5. The DHAlevel in ten of the 20 seeds analysed was in the range of 7-20% of thetotal fatty acid content as determined by the GC analysis. Other seedshad less than 7% DHA and may have contained a partial (incomplete) copyof the T-DNA from pJP3416-GA7-modB. The transgenic line appeared tocontain multiple transgene insertions that were genetically unlinked.The seeds of transgenic line CT133.15 exhibited DHA levels in the range0-5%. Seeds with no DHA were likely to be null segregants. These dataconfirmed that the modB construct performed well for DHA production incanola seed.

The pJP3416-GA7-modB and pJP3416-GA7-modF constructs were also used togenerate transformed Camelina sativa plants. At least 24 independenttransformed plants (T0) were obtained and examined in more detail byprogeny analysis. Seed (T1 seed) was harvested from these transgeniclines. Pools of seed were tested for levels of DHA in the seed oil, and6 lines which showed the highest levels of DHA (between 6% and 9%) wereselected. The DHA levels in 20 T1 seeds from each line wereanalysed-most seeds exhibited DHA levels in the range of 6-14% of thetotal fatty acid content as determined by the GC analysis. The fattyacid composition in the oil was determined; the data is shown in Table17 for several transgenic seeds. These data confirmed that the modB andmodF constructs both performed well for DHA production in Camelina seed.

TABLE 16 Fatty acid profiles of half cotyledons of germinating T1transgenic B. napus seeds containing the modB construct. Up to 18.1% DHAwas observed with numerous samples containing greater than 10% DHA. Seed14:0 16:0 16:1d3? 16:1 16:3 18:0 18:1 18:1d11 18:2 18:3n6 18:3n3 20:018:4n3 C20:1d11 20:1d13 1 0.1 4.2 0.1 0.1 0.2 1.8 29.9 2.5 9.9 0.1 38.40.5 0.8 1.0 0.0 2 0.1 4.7 0.1 0.1 0.2 4.0 23.0 2.3 7.4 0.3 29.3 1.0 4.31.1 0.0 3 0.1 3.7 0.2 0.1 0.2 1.8 55.1 1.9 4.7 0.2 15.2 0.8 1.8 1.4 0.04 0.1 4.6 0.2 0.2 0.2 2.9 22.1 1.8 6.6 0.4 26.5 1.0 7.2 1.0 0.0 5 0.14.0 0.1 0.1 0.2 1.7 27.4 2.1 8.1 0.3 26.4 0.6 2.8 1.0 0.0 6 0.1 3.5 0.10.1 0.2 1.6 59.8 2.0 4.3 0.1 18.5 0.6 0.5 1.3 0.0 7 0.1 6.0 0.3 0.3 0.31.7 16.6 2.6 23.9 1.0 23.2 0.6 5.4 0.8 0.0 8 0.1 4.9 0.1 0.1 0.2 2.712.9 1.4 11.7 0.3 34.3 0.9 5.0 0.9 0.0 9 0.1 3.9 0.1 0.1 0.1 2.4 41.61.7 21.5 0.0 23.4 0.7 0.0 1.2 0.0 10 0.1 3.7 0.2 0.1 0.1 2.1 30.9 1.719.2 0.4 23.6 0.7 2.1 1.1 0.0 11 0.1 5.7 0.4 0.3 0.2 3.8 41.2 2.4 26.72.1 7.2 1.3 0.3 1.2 0.0 12 0.1 4.6 0.0 0.1 0.2 2.4 25.5 1.7 16.1 0.328.9 0.8 3.9 1.1 0.0 13 0.1 4.3 0.1 0.1 0.1 4.2 19.4 1.6 9.2 0.1 45.51.0 0.2 1.1 0.0 14 0.1 6.3 0.2 0.2 0.2 4.0 10.5 2.3 8.4 0.3 31.1 1.3 3.90.8 0.0 15 0.1 5.1 0.1 0.2 0.2 3.3 16.8 2.4 11.2 0.3 28.8 1.0 4.5 0.90.0 16 0.1 4.4 0.1 0.1 0.2 4.0 16.2 1.5 11.6 0.2 33.5 0.9 2.8 1.1 0.0 170.2 7.2 0.2 0.2 0.2 4.9 15.0 2.1 8.9 0.3 25.9 1.4 5.1 0.9 0.0 18 0.1 4.00.1 0.1 0.2 2.3 64.8 1.2 7.2 0.1 12.5 1.0 3.5 1.5 0.0 19 0.1 3.9 0.1 0.10.2 4.6 36.9 1.7 7.1 0.2 28.6 1.2 1.8 1.2 0.0 20 0.1 4.8 0.1 0.1 0.2 6.018.5 1.2 12.8 0.2 34.8 1.4 2.4 1.1 0.0 Seed C20:2n6 C20:3n3 C22:0 20:4n320:5n3 22:3n3 C24:0 C24:1 22:5n3 C22:6n3 1 0.1 2.1 0.3 2.8 0.3 0.1 0.20.2 0.5 3.9 2 0.1 1.9 0.4 6.9 1.0 0.0 0.3 0.1 1.7 9.5 3 0.1 0.3 0.5 11.30.0 0.0 0.3 0.2 0.0 0.0 4 0.1 0.8 0.5 11.2 1.9 0.0 0.2 0.2 1.7 8.7 5 0.11.5 0.3 7.6 1.5 0.0 0.1 0.1 1.8 12.2 6 0.0 0.7 0.3 6.0 0.0 0.0 0.2 0.10.0 0.0 7 0.2 0.6 0.4 2.6 1.1 0.0 0.3 0.3 1.7 9.9 8 0.2 2.4 0.5 4.1 1.30.0 0.2 0.2 1.8 13.8 9 0.1 2.2 0.4 0.0 0.0 0.1 0.3 0.2 0.0 0.0 10 0.11.5 0.4 3.6 0.6 0.0 0.2 0.1 0.7 6.9 11 0.2 0.3 0.8 4.8 0.0 0.0 0.6 0.30.0 0.0 12 0.1 1.9 0.4 3.9 0.6 0.0 0.2 0.0 1.1 6.2 13 0.1 5.2 0.4 2.60.3 0.2 0.2 0.1 0.4 3.4 14 0.1 2.3 0.6 4.6 1.8 0.1 0.3 0.2 2.5 18.1 150.1 2.1 0.6 3.2 1.5 0.1 0.3 0.1 1.8 15.1 16 0.2 3.7 0.4 4.6 0.7 0.1 0.30.1 1.3 12.1 17 0.0 1.6 0.8 4.9 2.1 0.0 0.6 0.3 2.2 15.0 18 0.1 0.0 0.70.0 0.0 0.0 0.5 0.2 0.0 0.0 19 0.1 1.4 0.5 4.3 0.4 0.0 0.4 0.1 0.8 4.320 0.1 3.4 0.6 3.2 0.4 0.1 0.3 0.1 0.7 7.6

TABLE 17 Fatty acid profiles of T1 transgenic C. sativa seeds containingthe modB or modF constructs C14:0 C16:0 C16:1 C18:0 C18:1 C18:1d11 C18:2C18:3n6 C18:3n3 C20:0 123-8  0.1 7.3 0.0 5.2 7.9 1.0 7.7 0.7 29.9 2.3123-12  0.1 8.3 0.0 5.3 7.2 1.2 8.7 0.9 27.2 2.5 5-8 0.1 8.3 0.1 3.5 9.41.3 8.1 1.1 29.0 1.0 5-9 0.1 8.1 0.0 3.5 9.4 1.2 8.4 1.2 29.2 1.0 17-100.1 8.7 0.1 4.1 8.4 1.3 5.5 1.2 26.1 1.6 17-26 0.1 8.8 0.1 5.5 5.0 1.37.6 0.9 27.8 2.7 18:4n3 C20:1d11 20:1d13 C20:2n6 C20:3n6 C20:4n6 C20:3n3C22:0 20:4n3 123-8  6.0 7.1 0.4 0.7 0.0 0.0 0.9 0.4 1.3 123-12  5.7 6.90.5 0.7 0.0 0.1 0.9 0.5 1.5 5-8 9.3 7.9 0.4 0.6 0.0 0.0 0.8 0.2 0.4 5-99.0 8.1 0.3 0.6 0.0 0.0 0.8 0.2 0.5 17-10 11.8 7.2 0.3 0.0 0.4 0.03 0.80.3 0.4 17-26 10.1 6.2 0.3 0.0 0.7 0.03 1.1 0.6 0.5 C22:1 20:5n3 C22:2n622:3n3 C24:0 C24:1 22:5n3 C22:6n3 123-8  1.0 4.6 0.0 0.1 0.2 0.3 1.513.3 123-12  1.2 5.0 0.0 0.1 0.2 0.4 1.5 13.2 5-8 0.8 3.4 0.0 0.1 0.20.4 0.9 12.6 5-9 0.8 3.5 0.0 0.1 0.1 0.3 0.9 12.6 17-10 0.7 5.5 0.0 0.00.2 0.3 1.3 13.5 17-26 1.0 4.7 0.1 0.1 0.3 0.4 1.0 13.1

The inventors considered that, in general, the efficiency ofrate-limiting enzyme activities in the DHA pathway can be greater inmulticopy T-DNA transformants compared to single-copy T-DNAtransformants, or can be increased by inserting into the T-DNA multiplegenes encoding the enzyme which might be limiting in the pathway.Evidence for the possible importance of multi-copy transformants wasseen in the Arabidopsis seeds transformed with the GA7 construct(Example 2), where the highest yielding DHA event had three T-DNAsinserted into the host genome. The multiple genes can be identical, orpreferably are different variants that encode the same polypeptide, orare under the control of different promoters which have overlappingexpression patterns. For example, increased expression could be achievedby expression of multiple Δ6-desaturase coding regions, even where thesame protein is produced. In pJP3416-GA7-modF and pJP3416-GA7-modC, forinstance, two versions of the M. pusilla Δ6-desaturase were present andexpressed by different promoters. The coding sequences had differentcodon usage and therefore different nucleotide sequences, to reducepotential silencing or co-suppression effects but resulting in theproduction of the same protein.

Example 6. Activity of Seed-Specific Constructs in Somatic Embryos

In order to establish a rapid assay system which was predictive ofexpression of genetic constructs in seeds under the control ofseed-specific promoters, a somatic embryo system was set up for Brassicanapus. This used a vector to express the LEC2 transcription factor whichis involved in initiation of somatic embryogenesis. As a demonstration,the binary vectors 35S:LEC2 and pJP107 (Petrie et al., 2010a and b) wereintroduced into Agrobacterium tumefaciens strain AGL1 via standardelectroporation and the Agrobacterium transformants used to co-transformBrassica napus by co-cultivation. The T-DNA region of pJP107 containedgenes encoding the Isochrysis galbana Δ9-elongase, P. salinaΔ8-desaturase and P. salina Δ5-desaturase with each gene expressed by aseed-specific promoter. A control transformation used the 35S:LEC2vector alone. 35S:LEC2 expression resulted in the generation of somaticembryos in tissue culture directly from the transformed B. napus callustissue as described in Example 1.

Fatty acid analysis showed that the seed-specific genes on the T-DNA ofthe construct pJP107 were expressed in the transgenic somatic embryos inthe presence of the co-transformed LEC2 gene and functioned to produceARA (20:4^(Δ5,8,11,14)) from LA and EPA (20:5^(Δ5,8,11,14,17)) from ALA.The data for three co-transformed somatic embryos are shown in Table 18and the fatty acid composition of each compared to the fatty acidcomposition of seed oil from Brassica napus seed which was transgenicfor, and expressing, the T-DNA of pJP107 (Petrie et al., 2010a and b).Similar total percentages of ARA and the intermediate fatty acids EDA(20:2ω6) and DGLA (20:3ω6), as well as conversion efficiencies, wereobserved in somatic embryo tissue when compared with stably-transformedseed profiles. Similar results were observed in the fatty acidcompositions of the stable T₂ transgenic seed and somatic embryos: ω6fatty acids were at a level of 26.6% and 25.6% (on average),respectively, whilst ARA levels were found to be 9.7% and 10.6% (onaverage), respectively.

When 35S:LEC2 alone was introduced and the somatic embryos analysed in atime-course, the fatty acid profile was found to change to a moreembryo-like profile with 18:3^(Δ9,12,15) decreasing and 18:1^(Δ9)increasing in an inversely correlated manner (FIG. 8). These resultsindicated that the somatic embryos were indeed becoming seed-like incharacter and the genes on the T-DNA from pJP107 were expressed. Thisdemonstrated that the somatic embryo system allowed a rapidcharacterisation of transgenic seed-specific constructs in B. napuswithout requiring the full process of producing a transgenic plant and,from that, mature seed.

TABLE 18 Fatty acid composition of lipid obtained from Brassica napussomatic embryos generated by co-transforming pJP107 with 35S:LEC2,compared to the control untransformed (WT) and T₂ seeds transformed withpJP107. Individual enzymatic conversion efficiencies are shown inbrackets after the relevant enzymatic steps. D9-Elo is Δ9-elongase,D8-Des is Δ8-desaturase and D5-Des is Δ5-desaturase. WT T2 pJP107transgenic seed LEC2: #45 LEC2: #57 LEC2: #58 18:1^(Δ9) 57.2 45.7 3.82.5 1.9 18:2^(Δ9,12) 19.1 8.7 10 10.6 10 18:3^(Δ9,12,15) 10.2 4.1 22.527.5 24.2 20:2^(Δ11,14) 7.1 ± 1.9 (67% D9-elo)   5.2 (61.8% D9-elo)  3.7(56.7% D9-elo)  4.6 (61.8% D9-elo) 20:3^(Δ8,11,14) 1.1 ± 0.2 (60%D8-des) 0.4 (67% D8-des) 0.2 (73% D8-des)  0.4 (73% D8-des)20:4^(Δ5,8,11,14) 9.7 ± 0.9 (90% D5-des) 10.6 (98% D5-des)  10 (96%D5-des) 11.2 (97% D5-des)  20:3^(Δ11,14,17) 4.0 ± 0.8 9.9 5.5 7.320:4^(Δ8,11,14,17) 0.3 ± 0.1 0.4 0.3 0.4 20:5^(Δ5,8,11,14,17) 2.4 ± 0.27.6 6.4 7.9 Total new 24.6 34.1 26.1 31.8

Using the same system to generate somatic embryos, Brassica napus cellswere transformed separately with pJP3416-GA7-modB and pJP3416-GA7-modD.42 embryos were obtained, 18 for modB and 24 for modD. Total lipid wasextracted from the embryos and analysed for fatty acid composition. Theembryos contained between 0% and up to 16.9% DHA (Table 19). The resultswith 0% DHA was presumed to be due to integration of only a partialT-DNA or an insertion into a transcriptionally silent region of thegenome. The total ω3 FA (including ALA) to total ω6 FA (including LA)ratio was found to be 2.3 for embryo #270 and 11.96 for embryo #284. Thetotal ω6 FA (including LA) to total ω3 FA (including ALA) ratio was 0.08for #284. The new ω6 FA (excluding LA) to new ω3 FA (excluding ALA)ratio was 0.03 for #284. Overall conversion efficiencies were calculatedto be: (for embryos #270, #284) OA to EPA=14.0%, 29.8%; OA to DHA=9.7%,24.2%; LA to EPA=15.4%, 30.7%; LA to DHA=10.7%, 25.0%; ALA to EPA=22.1%,33.3%; ALA to DHA=15.3%, 27.0%. These efficiencies were similar, orgreater than in the case of #284, to those observed for the T₃pJP3416-GA7 Arabidopsis lines which indicated that the pJP3416-GA7-modBvector was capable of functioning well in B. napus cells. The SDA levelwas below 3.0%, indicating that the Δ6-elongase was performing evenbetter than the GA7 construct. The individual enzyme efficienciesachieved in #284 were: Δ12-desaturase, 97.4%; ω3-desaturase, 92.3%;Δ6-desaturase, 38.2%; Δ6-elongase, 88.2%; Δ5-desaturase, 98.8%;Δ5-elongase, 94.1%; and Δ4-desaturase, 86.3%. Total saturates were21.2%, total monounsaturates were 10.2%, total polyunsaturates were68.6%.

The inventors believe this was the highest level of DHA achieved in B.napus cells to date, except for further data described below. This alsodemonstrated that the modification in pJP3416-GA7-modB relative topJP3416-GA7 was effective in increasing the level of expression of theΔ6-desaturase gene. The binary vectors pJP3416-GA7, pJP3416-GA7-modA,pJP3416-GA7-modC, pJP3416-GA7-modD, pJP3416-GA7-modE andpJP3416-GA7-modF as described above are co-transformed with 35S:LEC2 togenerate transformed B. napus somatic embryos. Up to 7.0% DHA wasobserved in modD embryos, 9.9% in modE embryos, 8.3% in modF embryos and3.6% in a small number of modG embryos.

TABLE 19 Fatty acid composition of oil from Brassica napus somaticembryos #270 and #284 generated by co-transforming the seed-specific DHAacid construct pJP3416-GA7-modB with 35S:LEC2, and #286 and #289(pJP3416-GA7-modD). #270 #284 #286 #289 14:0 0.3 0.2 0.2 0.2 16:0 14.015.7 17.2 16.6 16:1d9 0.7 0.4 0.8 0.8 16:3 0.5 0.6 1.1 1.3 18:0 2.6 2.42.5 2.5 18:1d9 6.6 1.8 1.5 1.1 18:1d11 6.3 6.8 6.5 6.7 18:2 18.9 4.510.0 9.8 18:3ω6 0.7 0.8 0.3 0.3 18:3ω3 33.0 37.2 42.0 41.5 20:0 0.9 0.90.8 0.8 18:4ω3 1.9 2.8 3.6 4.5 20:1d11 0.2 0.1 0.1 0.1 20:2ω6 0.1 0.10.1 0.2 20:3ω3 0.5 0.0 0.5 0.6 22:0 0.8 1.5 0.6 0.7 20:4ω3 0.2 0.9 0.70.7 20:5ω3 0.7 0.2 0.3 0.3 22:2ω6 0.0 1.2 0.0 0.0 22:3ω3 0.0 0.1 0.0 0.124:0 0.8 1.0 1.0 1.0 24:1 0.8 1.0 0.7 0.9 22:5ω3 2.4 2.7 3.2 3.0 22:6ω37.0 16.9 6.1 6.4

Example 7. Analysis of TAG from Transgenic A. thaliana Seeds ProducingDHA

The positional distribution of DHA on the TAG from the transformed A.thaliana seed was determined by NMR. Total lipid was extracted fromapproximately 200 mg of seed by first crushing them under hexane beforetransferring the crushed seed to a glass tube containing 10 mL hexane.The tube was warmed at approximately 55° C. in a water bath and thenvortexed and centrifuged. The hexane solution was removed and theprocedure repeated with a further 4×10 mL. The extracts were combined,concentrated by rotary evaporation and the TAG in the extracted lipidpurified away from polar lipids by passage through a short silica columnusing 20 mL of 7% diethyl ether in hexane. Acyl group positionaldistributions on the purified TAG were determined quantitatively aspreviously described (Petrie et al., 2010a and b).

The analysis showed that the majority of the DHA in the total seed oilwas located at the sn-1/3 positions of TAG with little found at the sn-2position (FIG. 9). This was in contrast to TAG from ARA producing seedswhich demonstrated that 50% of the ARA (20:4^(Δ5,8,11,14)) was locatedat the sn-2 position of transgenic canola oil whereas only 33% would beexpected in a random distribution (Petrie et al., 2012).

Positional distribution of DHA in the TAG from the B. napus seedstransformed with pJP3416-GA7 or with the combination of pJP3115 andpJP3116 is determined by essentially the same method.

The total lipid from transgenic A. thaliana seeds was also analysed bytriple quadrupole LC-MS to determine the major DHA-containingtriacylglycerol (TAG) species (FIG. 10). The most abundantDHA-containing TAG species was found to be DHA-18:3-18:3 (TAG 58:12;nomenclature not descriptive of positional distribution) with thesecond-most abundant being DHA-18:3-18:2 (TAG 58:11). Tri-DHA TAG (TAG66:18) was observed in total seed oil, albeit at low but detectablelevels. Other major DHA-containing TAG species included DHA-34:3 (TAG56:9), DHA-36:3 (TAG 58:9), DHA-36:4 (TAG 58:10), DHA-36:7 (TAG 58:13)and DHA-38:4 (TAG 60:10). The identities of the two major DHA-containingTAG were further confirmed by Q-TOF MS/MS.

Example 8. Predicting DHA Production in B. napus Seeds

Efficient production of DHA in Arabidopsis seeds at a 15% level usingthe GA7 genetic construct was demonstrated in Example 2. The sameconstruct in Brassica napus seeds produced only about 1.5% DHA in many(but not all) of the transformants, primarily due to the poor expressionof the Δ6-desaturase gene of GA7 in this species (Example 4). Based onthe realisation that modifications to the GA7 construct would overcomethe Δ6-desaturase gene expression problem (see Example 5, asdemonstrated in Example 6), calculations were performed to determine thelikely fatty acid profile of B. napus transgenic seeds expressing thegenes from a variant of pJP3416-GA7, where each transgene-encoded enzymewas performing as efficiently as was observed in A. thaliana with theGA7 construct. The predicted fatty acid compositions for threecalculations (#1, #2, #3) are shown in Table 20. This was based on awild-type (non-transformed) fatty acid composition for B. napus thatincluded 59% oleic acid, 20% LA and 8% ALA. The three predicted partialfatty acid profiles shown in the lower half of the table were based onthe conversion efficiencies for each enzymatic step shown in the upperhalf of the table. In prediction #2, a combination of Δ12-desaturationat 75% efficiency, Δ15-desaturation at 75%, Δ6-desaturation at 35%,Δ6-elongation at 80%, Δ5-desaturation at 90%, Δ5-elongation at 90% andΔ4-desaturation at 90% would result in the production of approximately10% DHA in a typical canola transgenic seed. These efficiencies were alllower or about equal to the individual efficiencies seen in Arabidopsis,so prediction #2 represented a conservative estimate. The conversionefficiencies listed in #3 were approximations based on the efficientconversions seen in A. thaliana transformed with pJP3416-GA7. DHA waspredicted to be produced at about 15% of the total fatty acid content inseedoil produced in B. napus seed, a result that mirrored the mostefficient production levels observed in A. thaliana. Insertion ofmultiple T-DNAs in the homozygous state is expected to raise the DHAlevel to 20% in B. napus.

TABLE 20 Predicted fatty acid composition for selected fatty acids as apercentage of total fatty acid content in seedoil from Brassica napustransformed with a DHA pathway construct, based on observed enzymaticefficiencies in transgenic Arabidopsis. The enzymes are listed in orderin the pathway for producing DHA from oleic acid. des = desaturase, elo= elongase. Predicted fatty acid compositions #1, #2 and #3 are based onthe efficiencies in the upper half of the Table. Enzyme #1 #2 #3 d12-des70% 75% 80% d15-des 70% 75% 80% d6-des (ω3) 30% 35% 40% d6-elo 80% 80%90% d5-des 80% 90% 90% d5-elo 80% 90% 90% d4-des 80% 90% 90% Fatty acidWT #1 #2 #3 18:1d9 59% 26%  22%  18%  18:2ω6 20% 19%  17%  14%  18:3ω61% 2% 3% 18:3ω3  8% 30%  32%  34%  18:4ω3 3% 3% 2% 20:4ω3 2% 1% 2%20:5ω3 2% 1% 2% 22:5ω3 1% 1% 2% 22:6ω3 5% 10%  15% 

Example 9. Stable Expression of a Transgenic EPA Pathway in Plant LeafBinary Vector Construction

A binary vector, pORE04+11ABGBEC_Cowpea_EPA_insert (SEQ ID NO:8), wasdesigned for introduction of a T-DNA into plants for the synthesis ofEPA in leaf tissues. It contained chimeric genes encoding the enzymes:M. pusilla Δ6-desaturase (SEQ ID NO:16), P. cordata Δ6-elongase (SEQ IDNO:25) and P. salina Δ5-desaturase (SEQ ID NO:30), each under thecontrol of the CaMV 35S and A. thaliana rubisco small subunit (SSU)promoters (FIG. 9). The binary vector was constructed by synthesisingthe region 199-10878 of SEQ ID 2 and cloning this into the recipientbinary vector pORE04 (Coutu et al., 1997) at the BsiWI and KasI sites.The three fatty acid biosynthesis genes coded for the enzymes requiredto convert ALA, 18:3^(Δ9,12,15) to EPA, 20:5^(Δ5,8,11,14,17).

Transient Expression of EPA Construct in N. benthamiana Leaf Cells

To test that the construct was correct and would express the genesefficiently in leaf tissues, the chimeric vectorpORE04+11ABGBEC_Cowpea_EPA_insert was introduced into A. tumefaciensstrain AGL1. The chimeric vector 35S:p19 was also introduced into A.tumefaciens strain AGL1 as described in Example 1. Cells from culturesof these infiltrated into leaf tissue of Nicotiana benthamiana plants ina 24° C. growth room. Several direct comparisons were infiltrated withthe samples being compared located on either side of the same leaf.Experiments were performed in triplicate. Following infiltration, theplants were grown for a further five days before leaf discs were takenfor fatty acid profile analysis by GC as described in Example 1. GCanalysis revealed that the EPA vector was functioning to produce EPA inNicotiana benthamiana leaf (Table 21) with the highest level of EPAfound to be 10.7% of total leaf lipids.

Nicotiana tabacum Stable Transformation

The chimeric vector pOREO4+11ABGBEC_Cowpea_EPA insert was used to stablytransform Nicotiana tabacum. The vector was introduced into A.tumefaciens strain AGL1 via standard electroporation procedure. Thetransformed cells were grown on solid LB media supplemented withkanamycin (50 mg/L) and rifampicin (25 mg/L) and incubated at 28° C. fortwo days. A single colony was used to initiate fresh culture. Following48 h vigorous culture, the cells were collected by centrifugation at2,000×g and the supernatant was removed. The cells were resuspended infresh solution containing 50% LB and 50% MS medium at the density ofOD₆₀₀=0.5.

TABLE 21 Fatty acid composition of total leaf lipid from transgenicNicotiana benthamiana (transient) and Nicotiana tabacum (stable primarytransformant) events with the highest EPA levels from each experiment.N. benthamiana N. tabacum 14:0 0.1 0.1 16:0 18.5 17.8 16:1w13t 2.2 3.816:1d9 0.1 0 16:3 6.2 5.7 18:0 3.4 3.2 18:1d11 0.3 0.3 20:0 0.5 0.5 22:00.2 0.3 24:0 0.1 0.4 18:1 2.9 1.6 18:2ω6 12.6 14.5 Omega-6 18:3ω6 2.32.9 20:2ω6 0.0 0.0 20:3ω6 0.1 0.0 20:4ω6 0.3 0.7 Omega-3 18:3ω3 37.132.4 18:4ω3 1.6 1.9 20:3ω3 0.1 0.3 20:4ω3 0.3 1.1 20:5ω3 10.7 12.122:5ω3 0.3 0.4

Leaf samples of N. tabacum cultivar W38 grown in vitro were excised andcut into square sections around 0.5-1 cm² in size with a sharp scalpelwhile immersed in the A. tumefaciens solution. The wounded N. tabacumleaf pieces submerged in A. tumefaciens were allowed to stand at roomtemperature for 10 minutes prior to being blotted dry on a sterilefilter paper and transferred onto MS plates without supplement.Following a co-cultivation period of two days at 24° C., the explantswere washed three times with sterile, liquid MS medium, then blotted drywith sterile filter paper and placed on the selective MS agarsupplemented with 1.0 mg/L benzylaminopurine (BAP), 0.25 mg/Lindoleacetic acid (IAA), 50 mg/L kanamycin and 250 mg/L cefotaxime. Theplates were incubated at 24° C. for two weeks to allow for shootdevelopment from the transformed N. tabacum leaf pieces.

To establish rooted transgenic plants in vitro, healthy green shootswere cut off and transferred into 200 mL tissue culture pots containingMS agar medium supplemented with 25 μg/L IAA, 50 mg/L kanamycin and 250mg/L cefotaxime. Transgenic shoots were transferred to soil afterrooting and grown to maturity in the glasshouse. Sufficiently large leafdiscs were taken from 21 mature transgenic plants from and analysed forfatty acid profile as described in Example 1. All transgenic sampleswere found to contain EPA (Table 21) with the highest level of EPA in ahemizygous primary transformant found to be 12.1% of total leaf lipids.The leaf samples also contained a small amount (<0.5%) of DPA in theirlipid, which resulted from elongation of the EPA by a low level ofΔ5-elongation activity of the Δ6-elongase. The total ω3 FA (includingALA) to ω6 FA (including LA) ratio was found to be 2.7. Overallconversion efficiencies were calculated to be: OA to EPA=18.4%, LA toEPA=18.9%, ALA to EPA=25.9%. The production of 12.1% EPA is notableespecially since the events were hemizygous primary transformants. TheALA to EPA efficiency in particular is close to that observed in stableseed transformants. It is worth noting that the construct did notcontain a Δ12 or Δ15-desaturase to increase the conversion of OA and LAto ALA. Increased efficiencies would be expected with addition of theseactivities.

Seed from hemizygous transformants is being harvested and sown out togenerate homozygous plants.

Seed set in the top EPA lines appeared normal and seed from lines #10and #17 germinated well to establish the T₂ generation. The ratio of EPAto null (no EPA) lines indicated that event #28 was single-locus and theT₃ generation of this line was therefore also established. Fatty acidprofile analysis of the T₃ population indicated that the transgenes werehomozygous with no null events found and a stable amount of EPA. Theaverage amount of EPA in the total leaf lipids in the entire T₃population was found to be 9.4%+0.3 (Table 22).

TABLE 22 Representative fatty acid profiles of total leaf lipids fromwildtype (WT) and independent transgenic or transiently-transformedlines (EPA). Species are Nicotiana benthamiana (transienttransformation), N. tabacum (a stably transformed T₃ population), Vignaunguiculata (stably transformed T₁ event). The errors denote standarddeviation of multiple samples. Apparent conversion efficiencies shown atthe bottom describe the ω3 pathway and are calculated as the sum ofproduct FAs/sum of substrate + product FAs. N. benthamiana N. tabacum V.unguiculata WT EPA WT EPA WT EPA 16:0  17.7 ± 0.1 18.7 ± 0.2  15.0 ±0.6  16.5 ± 0.5  18.0 18.2 ± 0.2  16:1ω13t  3.2 ± 0.1 2.2 ± 0  3.5 ± 0.13.0 ± 0.3 3.8 2.0 ± 0.9 16:3  6.8 ± 0.1 6.2 ± 0.1 5.2 ± 0.5 5.4 ± 0.3 —— 18:0 3.1 ± 0 3.5 ± 0.3 2.2 ± 0.2 2.6 ± 0.1 1.8 4.5 ± 0.4 Minor 1.4 ± 01.4 ± 0.1 3.1 ± 0.4 2.5 ± 0.3 2.3 2.5 ± 0.4 OA  1.7 ± 0.1 2.7 ± 0.2 1.6± 0.3 2.1 ± 0.3 2.0 4.3 ± 1.3 LA  12.5 ± 0.4 12.7 ± 0.2  17.0 ± 1.1 18.0 ± 0.9  13.4 18.2 ± 3.0  ALA  53.3 ± 0.2 37.2 ± 0.2  52.2 ± 1.9 34.0 ± 0.6  58.6 38.2 ± 0   Omega-6 GLA — 2.3 ± 0.1 — 2.3 ± 0.3 — 0.6 ±0.2 20:2ω6 0.1 ± 0 — 0.1 ± 0  0.1 ± 0  — 0.1 ± 0  DGLA 0.1 ± 0 0.1 ± 0 — — — — ARA — 0.3 ± 0  — 0.7 ± 0.1 — 0.2 ± 0  Omega-3 SDA — 1.5 ± 0.1 —1.6 ± 0.1 — 1.5 ± 0  20:3ω3 0.1 ± 0 0.1 ± 0  0.1 ± 0  0.3 ± 0  0.1 ± 01.5 ± 0.1 ETA — 0.4 ± 0  — 1.1 ± 0.1 — 0.3 ± 0.2 EPA — 10.2 ± 0.5  — 9.4± 0.3 — 7.1 ± 0.2 DPA — 0.3 ± 0.1 — 0.4 ± 0  — 0.8 ± 0.1 Omega-3 Δ6-des25% 27% 20% conversion Δ6-elo 88% 87% 85% Δ5-des 97% 90% 96% Δ5-elo  3% 4% 10%

Leaf samples of homozygous T₃ N. tabacum plants were subjected tofurther biochemical analysis. Total lipids were extracted fromfreeze-dried leaf material and fractionated by thin-layer chromatography(TLC). EPA was found to be present in N. tabacum TAG at up to 30.1% aswell as in the polar lipids at 6.3% (Table 23). It was interesting tonote that the EPA produced by the transgenic pathway was present in allof the lipid fractions assessed including TAG, MGDG, DGDG, SQDG, PG, PC,PE, PI and PS. All lipid pools contained low levels of novelintermediate or ω6 LC-PUFA fatty acids with the TAG ratio of novel ω3 toω6 fatty acids being 10:1.

Stable Transformation of Cowpea

The chimeric vector pORE04+11ABGBEC-Cowpea-EPA-insert was transformedinto cowpea (Vigna unguiculata) as follows. Mature dry seeds are thepreferred starting material although seeds harvested from immature podsat maximum fresh weight of seeds can also be used. Dry seeds arethreshed by hand to avoid cracking of seed coats and thus reducecontamination with microorganisms.

Dry seeds or immature pods are submerged in 70% ethanol for 2 min andthen treated for 30 min in 20% commercial bleach (8.4 g/L sodiumhypochlorite final concentration). The seeds are then washed severaltimes with sterile water. Immature seeds are removed aseptically frompods while mature seeds are imbibed overnight. Two different explantscan be used for multiple shoot production, ie the embryonic axis and thecotyledon itself, preferably the cotyledon with the bisected embryonicaxis attached. The shoot and root tips are removed from the axis beforewounding at the cotyledonary node, i.e. the point of attachment of theaxis to the cotyledon. From an initial comparison of 19 cultivars andlines, it is now clear that most lines of cowpea can be transformed, theonly caveat being that different tissue culture conditions need to beoptimised for each line.

TABLE 23 Analysis of young and mature (young | mature) leaf lipidfractions triacylglycerol (TAG), total polar lipid (PL),monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG),sulfoquinovosyldiacylglycerol (SQDG), phosphatidylglycerol (PG),phosphatidylcholine (PC), phosphatidylethanolamine (PE),phosphatidylinositol (PI) and phosphatidylserine (PS) from transgenicNicotiana tabacum leaf samples. The errors denote standard deviation ofmultiple samples. Up to 30% EPA was observed in leaf TAG with EPA alsodistributed throughout the polar lipids. Differences between yound andmature leaf profiles were also observed for several fatty acids.Chloroplastidic Extra-chloroplastidic TAG PL MGDG DGDG SQDG PG PC PE PIPS 16:0  9.8|18.3 17.8|23.8 3.1|3.2 18.0|16.8 48.3|50.0 21.0|26.422.9|30.0 24.0|30.5 38.7|43.3 31.9|36.2 16:1ω13t 0|0 3.4|3.1 0|0 0|0 0|034.0|32.0 0 |0  0|0 0|0 1.0|1.4 16:3 0.2|0.9 5.6|6.4 14.8|19.4 1.2|1.80.4|1.2 0|0 0|0 0|0 0|0 0|0 18:0 7.3|3.7 2.9|3.9 1.1|1.2 3.5|3.5 5.4|7.14.7|6.9 6.6|9.1 11.0|11.4 9.4|9.3 20.2|19.4 Minor 2.5|2.9 1.4|2.41.0|0.4 0.8|1.0 1.9|2.1 1.0|1.5 1.4|1.6 4.9|4.1 6.5|7.7 2.5|3.7 OA5.5|0.8 2.8|1.1 0.8|0.3 1.8|1.0 2.7|1.3 5.3|4.9 8.1|2.9 2.5|1.1 2.5|0.84.9|2.3 LA 27.7|13.7 17.3|12.3 8.0|6.8  9.2|10.5 11.7|8.9  17.1|13.239.2|25.2 37.9|28.5 22.0|13.4 24.4|17.1 ALA  9.6|17.2 39.0|34.460.3|51.9 61.2|58.6 23.7|21.5 15.7|14.1  7.3|18.2  5.5|10.5  7.6|10.0 4.8|10.5 Omega-6 GLA 2.5|3.0 1.5|2.1 2.1|3.0 1.1|1.8 1.4|1.9 0.2|0 1.8|2.5 1.7|2.7 0.8|0.9 1.1|1.3 20:2ω6 0|0 0.1|1.1 0|0 0|0 0|0 0|0 0|00.5|0  0|0 0|0 DGLA 0|0 0|0 0|0 0|0 0|0 0|0 0|0 0|0 0|0 0|0 ARA 0.6|0.90.1|0.2 0.2|0.4 0|0 0|0 0|0 0.3|0.3 0.4|0.4 0.4|0.6  0|0.2 Omega-3 SDA4.0|7.6 1.6|2.0 1.7|2.0 0.6|0.7 1.2|1.2 0|0 2.1|3.6 1.3|2.0 0.8|0.80.9|1.6 20:3ω3 0.2|0.3 0.1|0.2 0 |0  0.2|0.3 0|0  0|0.1 0.2|0  0.3|0.40|0 0|0 ETA 0.9|0.2 0.2|0.3  0|0.2  0|0.3 0|0 0|0 0.2|0  0.4|0.2 0.1|0.20|0 EPA 28.8|30.1 6.1|6.3  6.9|11.2 2.3|3.6 3.4|4.6 1.0|0.8 9.7|6.49.1|7.8 11.2|12.8 8.4|6.2 DPA 0.4|0.5 0 |0   0|0.1 0|0 0|0 0|0 0.3|0.40.5|0.4  0|0.2  0|0.1

The selectable marker genes, bar or NptII can be used fortransformation. The Agrobacterium tumefaciens strain AGL1 is thepreferred strain for cowpea transformation. Agrobacterium containing thepOREO4+11ABGBEC-Cowpea-EPA-insert vector is cultured overnight at 28° C.on a shaker at 180 rpm and the suspension is centrifuged at 8000 g for10 min and re-suspended in Medium 1 (MS-basic medium diluted one in tenand containing 30 g/l sucrose, 20 mM 2-MES, adjusted to pH 5.6 prior toautoclaving, supplemented with filter sterilized MS-vitamins, 100 mg/lmyo-inositol, 1.7 mg/l BAP, 0.25 mg/l GA3, 0.2 mM acetosyringone, 250mg/l Na-thiosulphate, 150 mg/l dithiothreitol and 0.4 g/l L-cysteine).The explants are submerged without shaking in the bacterial suspensionfor one hour following wounding in the meristematic regions with ascalpel. The treated explants are then blotted on sterile filter paperand transferred to solidified Medium 2 (Medium 1 containing 0.8% agar)overlayed with filter paper. After four days of co-cultivation, explantsare transferred to Medium 3 (full strength MS medium, supplemented with100 mg/l myo-inositol, 150 mg/l timentin, 30 g/L sucrose, 3 mM MES, 1.7mg/L BAP, 5 mg/L PPT or 25-50 mg/L geneticin or 150 mg/L kanamycin, 0.8g/L agar and adjusted to pH 5.6) for shoot initiation and selection oftransformed shoots. After two weeks the first shoots are visible. Thecotyledons are removed from the cotyledonary node region and culturesare transferred to fresh Medium 3. Cultures are transferred to freshMedium 3 every two weeks following removal of dead and dying tissue. Thefirst four subcultures are on kanamycin selection followed byalternating with geneticin and kanamycin. After six sub-cultures, thesurviving green shoots are transferred to Medium 4 (Medium 3 without BAPbut supplemented with 0.5 mg/l GA3, 50 mg/l asparagine, 0.1 mg/l3-indoleacetic acid (IAA), 150 mg/l timentin, and either PPT (10 mg/1),geneticin (50 mg/L) or kanamycin (150 mg/L), for shoot elongation. Theshoots are sub-cultured every two weeks until single shoots are morethan 1 cm long. These larger shoots are transferred from petri dishes toculture jars (80 mm height) for further growth under selection.

The majority of the regenerated shoots can be rooted in vitro, and therooted plants are transferred to soil and allowed to establish in a highhumidity chamber for 14-21 days before transfer to ambient greenhouseconditions.

To enhance gene transfer to cowpea, co-culture media is supplementedwith thiol compounds. The addition of L-cysteine, dithiothreitol, andsodium thiosulfate reduces browning of wounded tissue.

Large numbers of cowpea explants can be processed in a simplifiedprotocol. In brief, the protocol consists of the following steps:imbibition of sterilized mature seeds overnight in water, explants arederived by longitudinally bisecting the seed as a result of which, thesplit embryonic axis (with shoot and root apices removed) is stillattached to the cotyledon, infection with Agrobacterium strain AGL1aided by local wounding in the meristematic regions, co-culture onmedium containing thiol compounds over 4 days at 25° C. in light, shootinitiation and elongation on medium containing selective agents, shootsare rooted in vitro and transferred to greenhouse conditions forflowering and seed setting, PCR or enzyme analysis of putativetransgenic plants, and screening of next generation progeny by PCR orenzyme activity.

The progeny of transgenic T₀ plants are normal in phenotype. Thetransgenes are transmitted to the progeny and homozygous T₂ plants areidentified by screening their T₃ progeny for enzyme activity or by PCR.

Using this transformation system about 10 transgenic plants are producedper 1000 explants, which is similar to the transformation frequency forother legumes. Depending on the cultivar or line to be transformed, thisprotocol requires 5-8 months from explant preparation to harvested T₁seeds.

The transformation system is used to introduce thepORE04+11ABGBEC-Cowpea-EPA-insert binary vector into regenerated,transformed cowpea plants.

Modifications to the pOREO4+11ABGBEC-Cowpea-EPA-insert binary vector aremade in which genes encoding a Δ5-elongase and Δ4-desaturase are added,to provide a genetic construct which confers the ability to furtherconvert the produced EPA to DHA. The construct is transformed intoplants for production of DHA in vegetative tissues.

EPA was found to be present in the small number of events survivingchemical selection. The highest line contained 7.1%+0.2 EPA in the totalleaf lipids. The rate of transformation was lower than usuallyexperienced for cowpea with only six lines confirmed transgenic. It is,as yet, unknown what caused this effect although it is interesting tonote that a larger than usual proportion of transgenic events containedincomplete T-DNA regions. It is possible that the large construct sizecontributed to the reduced efficiency. The apparent conversionefficiencies of each of the three transgenic enzymes were alsocalculated (Table 22). Results were broadly similar in all three specieswith good conversion to EPA after initial Δ6-desaturation of the nativeALA. Some Δ5-elongation of EPA to DPA was noted despite the absence of aspecific Δ5-elongase. The P. cordata Δ6-elongase has previously beenshown to have a low level of Δ9-elongase activity (i.e. 18:3^(Δ9,12,15)to 20:3^(Δ11,14,17) conversion) although no Δ5-elongase activity wasdetected in a yeast assay.

Example 10. Testing Variations of Δ12-Desaturase Genes Binary VectorConstruction

In an attempt to test and compare a series of chimeric Δ12-desaturasegenes, several binary vectors were made which were used to transform A.thaliana and B. napus. The binary vectors pJP3365, pJP3366, pJP3367,pJP3368 and pJP3369 each contained genes that encoded the P. pastorisω3-desaturase (SEQ ID NO: 12) and M. pusilla Δ6-desaturase (SEQ IDNO:16) enzymes, and one of a series of Δ12-desaturases. TheΔ12-desaturases were from Cryptococcus neoformans (Accession No.XP_570226 in pJP3365), a version of the Cryptococcus neoformansΔ12-desaturase which contained a L151M mutation in an attempt toincrease gene activity (in pJP3366), Lachancea kluyveri (SEQ ID NO:10 inpJP3367), Synechocystis PCC6803 (Accession No. BAA18169 in pJP3368) andCrepis palaestina (Accession No. CAA76157, Lee et al., 1998, inpJP3369). The Crepis desaturase was the only plant desaturase in theseries; the others were fungal enzymes. The vectors were made byinserting a plant codon-optimised protein coding region, except for theCrepis palestina Δ12-desaturase which was wildtype, for eachΔ12-desaturase into the NotI site of the vector pJP3364 (see FIG. 12),in the orientation operably linked to the FP1 promoter to provide forseed-specific expression of each desaturase. The vector pJP3364 alreadycontained the chimeric genes encoding the P. pastoris ω3-desaturase andM. pusilla Δ6-desaturase, each under the control of seed-specificpromoters (FIG. 12). The combination of the three fatty acidbiosynthesis enzymes, namely Δ12-desaturase, ω3-desaturase andΔ6-desaturase, was designed to assemble a pathway to convert oleic acid(18:1^(Δ9)) to SDA (18:4^(Δ6,9,12,15)). Assays were therefore carriedout to measure the level of SDA production in transformed seeds.

A. thaliana and B. napus Transformation and Analysis

The chimeric binary vectors were introduced into A. tumefaciens strainAGL1 and cells from cultures of the transformed Agrobacterium used totransform fad2 mutant A. thaliana plants using the floral dip method fortransformation (Clough and Bent, 1998). After maturation, the T₁ seedsfrom the treated plants were harvested and plated on MS platescontaining kanamycin for selection of plantlets having the NptIIselectable marker gene present on the T-DNA of each chimeric vector.Surviving T₁ seedlings were transferred to soil. After allowing theplants to self-fertilise and growing them to maturity, the T₂ seeds fromthese plants were harvested and the fatty acid composition of seedlipids analysed by GC.

The chimeric vector pJP3367 was also used to transform B. napus by themethod described in Example 4 to generate 12 transgenic events. SDA wasfound to range from 0.6% to 2.2% in pooled seed of the plants, and nineindividual seeds from the transgenic plant with the highest SDAtransgenic plant were analysed for fatty acid composition. Fatty acidcomposition data from such analysis is shown in Table 24.

The data showed that the Δ12-desaturase activity expressed from each ofthe T-DNAs in both A. thaliana and B. napus were unexpectedly low,providing an enzyme conversion efficiency of about 20% rather than the70-80% seen with the same expression cassette in the GA7 construct(Examples 2 and 3). The reason for this relatively poor expression ofthe Δ12-desaturase genes from these vectors is unclear but could berelated to the position of the genes in the construct as a whole.

In contrast, RT-PCR expression analysis demonstrated that the P.pastoris ω03-desaturase and M. pusilla Δ6-desaturase genes on the T-DNAswere relatively well expressed in the transformed seed. Table 24includes the Δ6-desaturase conversion efficiencies in the transformedseeds, which ranged from about 11% to about 25% in the one B. napustransformed line. This was considerably higher than the Δ6-desaturaseconversion efficiency of about 7% seen in the B. napus seeds transformedwith the GA7 construct (Example 4).

TABLE 24 Fatty acid composition as a percentage of total fatty acids inseed oil from single seeds from a T₁ Brassica napus plant transformedwith the T-DNA from pJP3367. SDA (18:4ω3) is shown in bold. SampleCT110-3#1 CT110-3#2 CT110-3#3 CT110-3#4 CT110-3#5 CT110-3#6 CT110-3#7CT110-3#8 CT110-3#9 C4:0 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 C6:0 4.34.2 4.1 4.5 3.8 4.3 4.0 5.0 4.7 16:1d7 0.1 0.1 0.1 0.1 0.0 0.1 0.1 0.10.1 C16:1d9 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.3 0.3 16:3 0.2 0.2 0.2 0.2 0.20.2 0.2 0.2 0.2 C18:0 1.9 1.9 1.3 1.8 2.1 1.8 2.4 3.1 2.2 C18:1 58.1 59.4  55.5  59.1  62.1  56.0  57.2  52.0  53.2  C18:1d11 3.5 3.6 3.0 3.22.9 3.6 3.2 4.4 3.5 C18:2 18.4  17.1  19.2  17.3  17.4  18.7  19.0 20.3  20.2  C18:3ω6 0.3 0.2 0.3 0.2 0.2 0.2 0.2 0.2 0.3 C18:3ω3 8.2 9.011.1  8.6 7.5 10.2  9.8 9.3 9.8 C20:0 0.5 0.5 0.4 0.5 0.6 0.5 0.6 0.70.6 18:4ω3 2.4 2.0 2.8 2.5 1.4 2.6 1.3 2.4 3.2 C20:1d11 1.1 1.1 1.2 1.21.2 1.1 1.2 1.1 1.1 20:liso  0.03  0.03  0.03  0.03  0.01  0.03  0.02 0.03  0.02 C20:2ω6 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 C22:0 0.2 0.20.2 0.2 0.2 0.2 0.2 0.3 0.2 C24:0 0.1 0.1 0.1 0.2 0.1 0.1 0.2 0.3 0.2C24:1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 Δ6-des % 22.9  17.9  20.3 22.8  15.8  20.2  11.7  20.9  24.9 

Therefore, to take advantage of the higher Δ6-desaturase conversionefficiencies conferred by the T-DNA from pJP3367, B. napus plantstransformed with this T-DNA were crossed to plants transformed with theT-DNA from pJP3416-GA7 (Example 4) to produce progeny plants and seedscarrying both T-DNAs. The fatty acid composition of oil extracted fromF1 seeds is analysed by GC for DHA content and other fatty acidcontents. Increased DHA levels are observed as a consequence ofincreased expression of the Δ6-desaturase. Plants which are homozygousfor both T-DNAs are produced and should produce higher levels of DHA.

Example 11. Increasing Accumulation of Fatty Acids by Using SilencingSuppressor Proteins Binary Vector Construction

WO 2010/057246 describes the use of silencing suppressor proteins (SSP)to increase transgene expression in the seeds of plants. To demonstratethat the use of such proteins could enhance and stabilise the productionof LC-PUFA in oilseeds over several generations, several SSP wereselected for testing, namely V2 (Accession No. GU178820.1), p19(Accession No. AJ288943.1), p38 (Accession No. DQ286869.1) and P0^(PE)(Accession No. L04573.1). p19 is a suppressor protein from Tomato BushyStunt Virus (TBSV) which binds to 21 nucleotide long siRNAs before theyguide Argonaute-guided cleavage of homologous RNA (Voinnet et al.,2003). V2, a suppressor protein from Tomato Yellow Leaf Curl Virus(TYLCV), binds to the plant protein SGS3 (Glick et al., 2008), a proteinthought to be required for the production of double stranded RNAintermediates from ssRNA substrates (Beclin et al., 2002), or binds todsRNA structures that have a 5′ overhangs (Fukunaga et al., 2009). p38is a suppressor protein from Turnip Crinkle Virus (TCV) which interfereswith plant silencing mechanisms by binding to Dicer and Argonauteproteins (Azevedo et al., 2010). P0 proteins such as P0^(PE) and RPV-P0,from poleroviruses, target Argonaut proteins for enhanced degradation(Baumberger et al., 2007; Bortolamiol et al., 2007, Fusaro et al.,2012). Genetic constructs were therefore prepared for expression ofthese SSP in plant seed in combination with a set of fatty acidbiosynthesis genes for production of ARA (20:4^(Δ5,8,11,14)) from LA(18:1^(Δ9,12)), as follows.

The fatty acid biosynthesis genes encoding the Isochrysis galbanaΔ9-elongase and the Pavlova salina Δ8- and Δ5-desaturases and thebacterial selection marker were obtained on a single DNA fragment frompJP3010 by digestion with PmeI and AvrII giving rise to a 9560 bpfragment. The Δ9-elongase coding region on this fragment was joined toan A. thaliana FAE1 promoter (pAtFAE1) and a conlinin transcriptiontermination/polyadenylation region (LuCnl2-3′). The desaturase codingregions were each joined to a truncated napin FP1 promoter (pBnFP1) anda nos3′ transcription termination/polyadenylation region. The threefatty acid biosynthesis genes on this fragment were oriented and spacedin the same manner as in pJP107 (Petrie et al., 2012) and encoded thesame proteins as pJP107. The DNA fragment also comprised apFP1:GFiP:nos3′ gene from pCW141 (see WO2010/057246) which encoded agreen fluorescent protein (GFP). This screenable marker gene was used asa visual seed-specific marker, allowing simple and non-destructiveidentification and thereby selection of transgenic seed comprising andexpressing the gene.

The PmeI-AvrII fragment was inserted into the PmeI-AvrII site of each ofa series of five vectors, each containing a different SSP gene(WO2010/057246), resulting in the genetic constructs designated pFN045,pFN046, pFN047, pFN048 and pFN049. These comprise the genes encoding theSSPs P0^(PE), p38, p19, 35S:V2 and V2, respectively. Each of the SSPgenes was under the control of the FP1 promoter and ocs3′ transcriptiontermination/polyadenylation region except in the construct pFN048 wherethe V2 coding region was under the control of the constitutive CaMV 35Spromoter. The SSP gene in each case was within the T-DNA region of theconstructs, adjacent to the right border (RB) of the T-DNA. A sixthconstruct, pFN050 which lacked any SSP coding sequence, was made bydigesting pFN045 with AhdI and NheI followed by recircularisation withDNA ligase to delete the FP1:P0^(PE) gene. Each of the six constructscomprised an NptII selectable marker gene within the T-DNA and adjacentto the left border of the T-DNA. All of the constructs had an RK2 originof replication for maintenance of the plasmids in Agrobacterium.

Transformation of A. Thaliana with ARA Expression Vectors in Combinationwith SSPs

To transform the genotype MC49 of Arabidopsis, which is afad2/fae1double mutant with high linoleic acid levels in its seed lipid, plantswere treated by the floral dip method (Clough and Bent, 1998) with A.tumefaciens strain GV3101 separately transformed with each of the sixconstructs pFN045-pFN050. The treated plants were grown to maturity andT₁ seeds harvested from them were plated on MS media containingkanamycin to select for transformed T₁ plants. Screening for GFPexpression in the seed was also used as a visual marker for transformedT₁ seeds. The seedlings which survived on MS/Kan plates or which wereobtained from GFP-positive seeds were transferred to soil and grown tomaturity for T₂ seeds. The numbers of transformed plants obtained were5, 14, 32, 8, 23 and 24 for the transformations with pFN045, pFN046,pFN047, pFN048, pFN049 and pFN050, respectively. It was discovered atthis stage that the gene encoding p38 in pFN046 was not functional andtherefore plants transformed with the vector pFN046 were considered asadditional controls i.e. essentially the same as for pFN050.

About 100 pooled T₂ seeds were taken from each transformed plant forfatty acid composition determination of seed lipid by FAME preparationand GC analysis. Six T₂ seedlings from each transgenic line were alsogrown to produce T₃ seeds.

The fatty acid composition in total lipid extracted from the T₂ seedswas determined using GC. The analysis showed a range of levels of ARAand the intermediates EDA (20:2ω6) and DGLA (20:3ω6) in the T₂populations. The data for ARA is shown in FIGS. 13 and 14.

FIG. 13 shows a box-plot analysis of the ARA level in the lipid of thepopulations of the T₂ seeds. It was evident that the median (50^(th)percentile) level of ARA in the populations of seeds which contained theFP1:p19 and 35S:V2 genes in addition to the ARA biosynthetic genes wassignificantly higher than in seeds containing the defective FP1:p38 geneor the control T-DNA from pFP050 which did not contain an SSP gene. Theaverage ARA levels for seeds transformed with genes encoding p19 and V2were greater than for seeds transformed with the p38 gene or thosewithout an SSP (FIG. 14). One FP1:p19 and two FP1:V2 lines exhibitedabout 19%, 20% and 23% ARA, respectively. These were outliers andtherefore not included in the calculations for the box-plot analysis.Fewer plants transformed with the T-DNAs comprising the genesFPI:P0^(PE) and 35S:V2 survived compared to the other constructs; it isthought that these genes could be detrimental to plant health in theMC49 background.

Not only were the ARA levels significantly different among theconstructs, the levels in seed lipid of the first intermediate of thepathway from LA to ARA, namely EDA (20:2ω6), was observed to be lower inlines expressing either V2 or p19 than in seeds lacking an SSP orcontaining the p38 construct (FIG. 15). In T₃ seeds, one populationcontaining the construct expressing p19 exhibited 38% ARA as apercentage of total fatty acids in the seed lipid.

A range of transgenic T₃ lines were progressed to the T₄ generation. Thelevels of ARA in the T₄ seeds expressing V2 were either the same ascompared to the previous generation, or indeed exhibited increasedlevels compared to their T₃ parents (FIG. 16). The lines expressing p19showed more varied ARA levels. The ARA level was decreased in some lineswhile in others it was the same or increased compared to the T3 parents.In contrast, the lines containing the defective p38 gene or lacking anSSP generally showed a decline in the level of ARA and an increase inthe levels of intermediates (FIG. 18). In some of these lines, ARA wasreduced to about 1% and levels of EDA had increased to about 20%. Themean levels of ARA in T₄ seeds were higher for lines expressing p19 andV2 compared to lines expressing p38 or lacking an SSP (FIG. 17).

This experiment showed that the expression of an SSP in seeds of atransgenic plant along with additional genes for a LC-PUFA biosyntheticpathway not only increased the level of production of the desired fattyacid in the first generation of progeny, but also stabilised the levelof the fatty acid production in later generations such as the third orfourth generation of progeny. The increased fatty acid production wasaccompanied by decreased levels of intermediate fatty acids in thebiosynthetic pathway. The SSP's p19 and V2 expressed from seed-specificpromoters were preferred. The construct designed to express the p38 SSPwas defective and no useful data were obtained with this construct. TheV2 SSP and its homologs from other viruses are thought to beparticularly preferred because they allow maximal expression of thebiosynthetic pathway genes and the simultaneous silencing of other genesin the same cells in the developing seed.

Example 12. Assaying Sterol Content and Composition in Oils

The phytosterols from 12 vegetable oil samples purchased from commercialsources in Australia were characterised by GC and GC-MS analysis asO-trimethylsilyl ether (OTMSi-ether) derivatives as described inExample 1. Sterols were identified by retention data, interpretation ofmass spectra and comparison with literature and laboratory standard massspectral data. The sterols were quantified by use of a5β(H)-Cholan-24-ol internal standard. The basic phytosterol structureand the chemical structures of some of the identified sterols are shownin FIG. 19 and Table 25.

The vegetable oils analysed were from: sesame (Sesamum indicum), olive(Olea europaea), sunflower (Helianthus annus), castor (Ricinuscommunis), canola (Brassica 20 napus), safflower (Carthamus tinctorius),peanut (Arachis hypogaea), flax (Linum usitatissimum) and soybean(Glycine max). In decreasing relative abundance, across all of the oilsamples, the major phytosterols were: β-sitosterol (range 28-55% oftotal sterol content), Δ5-avenasterol (isofucosterol) (3-24%),campesterol (2-33%), Δ5-stigmasterol (0.7-18%), Δ7-stigmasterol (1-18%)and Δ7-avenasterol (0.1-5%). Several other minor sterols wereidentified, these were: cholesterol, brassicasterol, chalinasterol,campestanol and eburicol. Four C29:2 and two C30:2 sterols were alsodetected, but further research is required to complete identification ofthese minor components. In addition, several other unidentified sterolswere present in some of the oils but due to their very low abundance,the mass spectra were not intense enough to enable identification oftheir structures.

The sterol contents expressed as mg/g of oil in decreasing amount were:canola oil (6.8 mg/g), sesame oil (5.8 mg/g), flax oil (4.8-5.2 mg/g),sunflower oil (3.7-4.1 mg/g), peanut oil (3.2 mg/g), safflower oil (3.0mg/g), soybean oil (3.0 mg/g), olive oil (2.4 mg/g), castor oil (1.9mg/g). The % sterol compositions and total sterol content are presentedin Table 26.

TABLE 25 IUPAC/systematic names of identified sterols. Sterol No. Commonname(s) IUPAC/Systematic name 1 cholesterol cholest-5-en-3β-ol 2brassicasterol 24-methylcholesta-5,22E-dien- 3β-ol 3chalinasterol/24-methylene 24-methylcholesta-5,24(28)E- cholesteroldien-3β-ol 4 campesterol/24-methyl- 24-methylcholest-5-en-3β-olcholesterol 5 campestanol/24-methyl- 24-methylcholestan-3β-olcholestanol 7 Δ5-stigmasterol 24-ethylcholesta-5,22E-dien- 3β-o l 9ergost-7-en-3β-ol 24-methylcholest-7-en-3β-ol 11 eburicol4,4,14-trimthylergosta-8,24(28)- dien-3β-ol 12 β-sitosterol/24-24-ethylcholest-5-en-3β-ol ethylcholesterol 13D5-avenasterol/isofucosterol 24-ethylcholesta-5,24(28)Z-dien- 3β-ol 19D7-stigmasterol/stigmast- 24-ethylcholest-7-en-3β-ol 7-en-3b-ol 20D7-avenasterol 24-ethylcholesta 7,24(28)-dien- 3β-ol

Among all the seed oil samples, the major phytosterol was generallyβ-sitosterol (range 30-57% of total sterol content). There was a widerange amongst the oils in the proportions of the other major sterols:campesterol (2-17%), Δ5-stigmasterol (0.7-18%), Δ5-avenasterol (4-23%),Δ7-stigmasterol (1-18%). Oils from different species had a differentsterol profile with some having quite distinctive profiles. In the caseof canola oil, it had the highest proportion of campesterol (33.6%),while the other species samples generally had lower levels, e.g. up to17% in peanut oil. Safflower oil had a relatively high proportion ofΔ7-stigmasterol (18%), while this sterol was usually low in the otherspecies oils, up to 9% in sunflower oil. Because they were distinctivefor each species, sterol profiles can therefore be used to help in theidentification of specific vegetable or plant oils and to check theirgenuineness or adulteration with other oils.

TABLE 26 Sterol content and composition of assayed plant oils. SterolSterol common Sun- Sun- Saf- Saf- Flax Flax Soy- number* name SesameOlive flower flower Castor Canola flower flower Peanut (linseed)(linseed) bean cold- cold- pressed pressed 1 cholesterol 0.2 0.8 0.2 0.00.1 0.3 0.2 0.1 0.2 0.4 0.4 0.2 2 brassicasterol 0.1 0.0 0.0 0.0 0.3 0.10.0 0.0 0.0 0.2 0.2 0.0 3 chalinasterol/24- 1.5 0.1 0.3 0.1 1.1 2.4 0.20.1 0.9 1.5 1.4 0.8 methylene cholesterol 4 campesterol/24- 16.2 2.4 7.47.9 8.4 33.6 12.1 8.5 17.4 15.7 14.4 16.9 methylcholesterol 5campestanol/24- 0.7 0.3 0.3 0.1 0.9 0.2 0.8 0.8 0.3 0.2 0.2 0.7methylcholestanol 6 C29:2* 0.0 0.0 0.1 0.2 0.0 0.1 0.5 0.5 0.0 1.2 1.30.1 7 Δ5-stigmasterol 6.4 1.2 7.4 7.2 18.6 0.7 7.0 4.6 6.9 5.1 5.8 17.68 unknown 0.5 1.3 0.7 0.6 0.8 0.7 0.7 1.3 0.4 0.7 0.6 1.3 9ergost-7-en-3β-ol 0.1 0.1 1.9 1.8 0.2 0.4 2.7 4.0 1.4 1.4 1.4 1.0 10unknown 0.0 1.3 0.9 0.8 1.2 0.9 1.8 0.7 1.2 0.7 0.5 0.7 11 eburicol 1.61.8 4.1 4.4 1.5 1.0 1.9 2.9 1.2 3.5 3.3 0.9 12 β-sitosterol/24- 55.345.6 43.9 43.6 37.7 50.8 40.2 35.1 57.2 29.9 28.4 40.2 ethylcholesterol13 Δ5-avenasterol/ 8.6 16.9 7.2 4.1 19.3 4.4 7.3 6.3 5.3 23.0 24.2 3.3isofucosterol 14 triterpenoid 0.0 2.4 0.9 1.1 0.0 0.0 1.6 1.9 0.0 0.00.0 0.9 alcohol 15 triterpenoid 0.0 0.0 0.7 0.6 0.0 0.0 2.8 1.8 0.0 0.00.3 0.0 alcohol 16 C29:2* 0.0 0.5 0.7 0.7 1.5 1.2 2.8 1.9 2.0 1.0 0.70.5 17 C29:2* 1.0 0.9 2.3 2.4 0.6 0.4 1.3 1.9 0.9 1.0 1.0 1.0 18 C30:2*0.0 0.0 0.0 0.0 1.9 0.0 0.0 0.0 0.0 0.0 0.0 0.0 19 Δ7-stigmasterol/ 2.27.1 9.3 10.9 2.3 0.9 10.5 18.3 1.1 7.9 8.7 5.6 stigmast-7-en-3β- ol 20Δ7-avenasterol 1.3 0.1 4.0 3.6 0.6 0.2 2.0 4.7 0.7 0.4 0.4 0.6 21unknown 0.7 7.1 0.9 0.8 0.0 0.4 0.3 0.4 0.0 3.0 3.6 0.0 22 unknown 0.30.0 0.3 0.9 0.0 0.0 1.2 1.3 0.2 0.1 0.0 0.3 23 unknown 0.2 0.2 0.3 0.30.2 0.1 0.3 0.2 0.2 0.1 0.2 0.5 24 unknown 0.0 3.1 0.9 1.3 0.6 0.4 0.20.4 0.7 1.7 1.9 0.8 25 unknown 0.9 0.4 0.3 0.5 0.3 0.1 0.5 0.7 0.3 0.10.1 0.6 26 C30:2 2.2 6.0 4.6 5.7 1.4 0.6 1.0 1.2 1.2 1.2 1.1 5.2 27unknown 0.0 0.4 0.4 0.3 0.3 0.2 0.1 0.2 0.3 0.1 0.0 0.3 Sum 100.0 100.0100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 Total sterol5.8 2.4 4.1 3.7 1.9 6.8 3.2 3.0 3.2 4.8 5.2 3.0 (mg/g oil) C29:2* andand C30:2* denotes a C29 sterol with two double bonds and a C30 sterolwith two double bonds, respectively

Two samples each of sunflower and safflower were compared, in each caseone was produced by cold pressing of seeds and unrefined, while theother was not cold-pressed and refined. Although some differences wereobserved, the two sources of oils had similar sterol compositions andtotal sterol contents, suggesting that processing and refining hadlittle effect on these two parameters. The sterol content among thesamples varied three-fold and ranged from 1.9 mg/g to 6.8 mg/g. Canolaoil had the highest and castor oil the lowest sterol content.

Example 13. Increasing Accumulation of DHA at the Sn-2 TAG Position

The present inventors considered that DHA accumulation at the sn-2position in TAG could be increased by co-expressing an1-acyl-glycerol-3-phosphate acyltransferase (LPAAT) together with theDHA biosynthesis pathway such as conferred by the GA7 construct or itsvariants. Preferred LPAATs are those which can act on polyunsaturatedC22 fatty acyl-CoA as substrate, resulting in increased insertion of thepolyunsaturated C22 chain at the sn-2 position of LPA to form PA,relative to the endogenous LPAAT. Cytoplasmic LPAAT enzymes oftendisplay varied substrate preferences, particularly where the speciessynthesises and accumulates unusual fatty acids in TAG. A LPAAT2 fromLimnanthes douglasii was shown to use erucoyl-CoA (C22:1-CoA) as asubstrate for PA synthesis, in contrast to an LPAAT1 from the samespecies that could not utilise the C22 substrate (Brown et al., 2002).

Known LPAATs were considered and a number were selected for testing,including some which were not expected to increase DHA incorporation atthe sn-2 position, as controls. The known LPAATs included: Arabidopsisthaliana LPAAT2: (SEQ ID NO: 63, Accession No. ABG48392, Kim et al.,2005), Limnanthes alba LPAAT (SEQ ID NO: 64, Accession No. AAC49185,Lassner et al., 1995), Saccharomyces cerevisiae Slc1p (SEQ ID NO: 65,Accession No. NP_010231, Zou et al., 1997), Mortierella alpina LPAAT1(SEQ ID NO: 67, Accession No. AED33305; U.S. Pat. No. 7,879,591) andBrassica napus LPAATs (SEQ ID NO: 68 and SEQ ID NO:69, Accession NosADC97479 and ADC97478 respectively). These were chosen to cover threegroups of LPAAT enzymes: 1) control plant seed LPAATs with typically lowactivity on unusual long-chain polyunsaturated fatty acids (includingthe Arabidopsis and Brassica LPAATs), 2. LPAATs that had previously beendemonstrated to act on C22 fatty acids by using C22 acyl-CoA assubstrate, in this case erucic acid C22:1 (including the Limnanthes andSaccharomyces LPAATs), 3. LPAATs which the inventors considered likelyto be able to utilise long-chain polyunsaturated fatty acids such as EPAand DHA as substrates (including the Mortierella LPAAT).

The Arabidopsis LPAAT2 (also designated LPAT2) is an endoplasmicreticulum-localised enzyme shown to have activity on C16 and C18substrates, however activity on C20 or C22 substrates was not tested(Kim et al., 2005). Limnanthes alba LPAAT2 was demonstrated to insert aC22:1 acyl chain into the sn-2 position of PA, although the ability touse DHA as a substrate was not tested (Lassner et al., 1995). Theselected S. cerevisiae LPAAT Slc1p was shown to have activity using22:1-CoA in addition to 18:1-CoA as substrates, indicating a broadsubstrate specificity with respect to chain length (Zou et al., 1997).Again, DHA-CoA and other LC-PUFAs were not tested as substrates. TheMortierella LPAAT had previously been shown to have activity on EPA andDHA fatty acid substrates in transgenic Yarrowia lipolytica (U.S. Pat.No. 7,879,591).

Additional LPAATs were identified by the inventors. Micromonas pusillais a microalga that produces and accumulates DHA in its oil, althoughthe positional distribution of the DHA on TAG in this species has notbeen confirmed. The Micromonas pusilla LPAAT (SEQ ID NO: 66, AccessionNo. XP_002501997) was identified by searching the Micromonas pusillagenomic sequence using the Arabidopsis LPAAT2 as a BLAST query sequence.Several candidate sequences emerged and the sequence XP_002501997 wassynthesised for testing as a likely LPAAT enzyme with activity on C22LC-PUFA. The Ricinus communis LPAAT was annotated as a putative LPAAT inthe castor genome sequence (Chan et al., 2010). Four candidate LPAATsfrom the castor genome were synthesised and tested in crude leaf lysatesof infiltrated N. benthamiana leaf tissue. The candidate sequencedescribed here showed LPAAT activity.

A number of candidate LPAATs were aligned with known LPAATs on aphylogenetic tree (FIG. 20). It was noted that the putative MicromonasLPAAT did not cluster with the putative C22 LPAATs but was a divergentsequence.

As an initial test of various LPAATs for their ability to use DHA-CoA assubstrate, chimeric genetic constructs are made for constitutiveexpression of exogenous LPAATs in N. benthamiana leaves, each under thecontrol of the 35S promoter, as follows: 35S:Arath-LPAAT2 (ArabidopsisER LPAAT); 35S:Ricco-LPAAT2; 35S:Limal-LPAAT (Limnanthes alba LPAAT);35S:Sacce-Slc1p (S. cerevisiae LPAAT); 35S:Micpu-LPAAT (Micromonaspusilla LPAAT); 35S:Moral-LPAAT1 (Mortierella alpina LPAAT). A 35S:p19construct lacking an exogenous LPAAT is used as a control in theexperiment. Each of these constructs is introduced via Agrobacteriuminto N. benthamiana leaves as described in Example 1, and 5 days afterinfiltration, the treated leaf zones are excised and ground to make leaflysates. Each lysate includes the exogenous LPAAT as well as theendogenous enzymes for synthesizing LPA. In vitro reactions are set upby separately adding ¹⁴C-labelled-OA, -LA or -ALA (C18 substrates), -ARA(a C20 substrate) and -DHA (C22) to the lysates, in triplicate.Reactions are incubated at 25° C. and the level of incorporation of the¹⁴C labelled fatty acids into PA determined by TLC. The ability of eachLPAAT to use DHA relative to ARA and the C18 fatty acids is calculated.The meadowfoam, Mortierella and Saccharomyces LPAATs were found to haveactivity on DHA substrate, with radiolabelled PA appearing for these butnot the other LPAATs. All LPAATs were confirmed active by a similaroleic acid feed.

To test LPAAT activity in seeds, several of the protein coding sequencesor LPAATs are inserted into a binary vector under the control of aconlinin (pLuCnl1) promoter. The resultant genetic constructs containingthe chimeric genes, Cnl1:Arath-LPAAT (negative control),Cnl1:Limal-LPAAT, Cnl:Sacce-Slc1p, and Cnl1:Moral-LPAAT, respectively,are then used transform B. napus and A. thaliana plants to generatestable transformants expressing the LPAATs in a seed-specific manner.The transformed plants having the Cnl1:LPAAT constructs are crossed withplants expressing the GA7 construct or its variants (Example 5) whichproduce DHA in the seed to result in increased incorporation of DHA atthe sn-2 position of TAG. The constructs are also used to transform B.napus, C. sativa and A. thaliana plants that already contain the GA7construct and variants thereof (Examples 2 to 5) to generate progenycarrying both the parental and LPAAT genetic constructs. Increasedincorporation of DHA at the sn-2 position of TAG is expected relative tothe incorporation in plants lacking the LPAAT encoding transgenes. Oilcontent is also improved in the seeds, particularly for seeds producinghigher levels of DHA, counteracting the trend seen in Arabidopsis seedas described in Example 2.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

The present application claims priority from U.S. 61/660,392 filed 15Jun. 2012, U.S. 61/663,344 filed 22 Jun. 2012, U.S. 61/697,676 filed 6Sep. 2012 and U.S. 61/782,680 filed 14 Mar. 2013, the entire contents ofeach of which are incorporated herein by reference.

All publications discussed and/or referenced herein are incorporatedherein in their entirety.

This application incorporates herein by reference U.S. 61/660,392 filed15 Jun. 2012, U.S. 61/663,344 filed 22 Jun. 2012 and U.S. 61/697,676filed 6 Sep. 2012.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed before the priority dateof each claim of this application.

REFERENCES

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1-85. (canceled)
 86. A process for producing extracted plant lipidcomprising the steps of: i) obtaining a Brassica sp. seed comprisingextractable lipid having a total fatty acid content comprising fattyacids in an esterified form, the fatty acids comprising oleic acid,palmitic acid, a total ω6 fatty acid content which comprises linoleicacid (LA) and γ-linolenic acid (GLA), a total ω3 fatty acid contentwhich comprises α-linolenic acid (ALA), stearidonic acid (SDA),docosapentaenoic acid, (DPA) and docosahexaenoic acid (DHA), andoptionally one or more of eicosapentaenoic acid (EPA) andeicosatetraenoic acid (ETA), wherein a) EPA, DPA and DHA, and optionallyARA, are each present at a level in the total fatty acid content, eachlevel being expressed as a percentage of the total fatty acid content,whereby the sum total of the levels of ARA, EPA, DPA and DHA in theextractable lipid is between 7% and 25% of the total fatty acid content,b) eicosatrienoic acid (ETrA) is present in the extractable lipid at alevel of between 0.05% and 3% of the total fatty acid content, c)palmitic acid is present in the extractable lipid at a level of between2% and 16% of the total fatty acid content, d) oleic acid is present inthe extractable lipid at a level of between 1% and 30% of the totalfatty acid content, e) LA is present in the extractable lipid at a levelof between 4% and 35% of the total fatty acid content, f) GLA is presentin the extractable lipid at a level of less than 4% of the total fattyacid content, g) ALA is present in the extractable lipid at a level ofbetween 4% and 40% of the total fatty acid content, h) the totalsaturated fatty acid content of the extractable lipid is between 4% and25% of the total fatty acid content, i) the ratio of the total 06 fattyacid content to the total ω3 fatty acid content of the extractable lipidis between 0.1 and 3.0, and j) myristic acid, if present, is present inthe extractable lipid at a level of less than 1% of the total fatty acidcontent. ii) extracting lipid from the Brassica sp. seed to therebyproduce extracted plant lipid.
 87. The process of claim 86, whereinγ-linolenic acid (GLA) is present in the extractable lipid at a level ofless than 2% of the total fatty acid content.
 88. The process of claim86, wherein oleic acid is present in the extractable lipid at a level ofbetween 3% and 30% of the total fatty acid content.
 89. The process ofclaim 86, wherein the total saturated fatty acid content of theextractable lipid is between 6% and 20% of the total fatty acid content.90. The process of claim 86, wherein the extractable lipid comprises new03 fatty acids in the total ω3 fatty acid content and new 06 fatty acidsin the total 06 fatty acid content, wherein the ratio of the new 06fatty acids to the new ω3 fatty acids in the extracted oil is between0.1 and
 1. 91. The process of claim 86, wherein the extractable lipidcomprises tri-DHA TAG (TAG 66:18).
 92. The process of claim 86, whereinat least 80% of the DHA esterified in the form of triacylglycerol (TAG)is at the sn-1 or sn-3 position of the TAG.
 93. The process of claim 86,wherein SDA, ETA, EPA, DPA and DHA are each present at a level in thetotal fatty acid content, each level being expressed as a percentage ofthe total fatty acid content, whereby the sum of the percentages forETA, EPA, DPA and DHA divided by the sum of the percentages for SDA,ETA, EPA, DPA and DHA, expressed as a percentage, is at least 75%. 94.The process of claim 86, wherein ETA, EPA, DPA and DHA are each presentat a level in the total fatty acid content, each level being expressedas a percentage of the total fatty acid content, whereby the sum of thepercentages for EPA, DPA and DHA divided by the sum of the percentagesfor ETA, EPA, DPA and DHA, expressed as a percentage, is between 70% and88%.
 95. The process of claim 86, wherein DHA is present in theextractable lipid at a level of about 3% of the total fatty acidcontent.
 96. The process of claim 86, wherein DHA is present in theextractable lipid at a level of about 4% of the total fatty acidcontent.
 97. The process of claim 86, wherein DHA is present in theextractable lipid at a level of about 5% of the total fatty acidcontent.
 98. The process of claim 86, wherein DHA is present in theextractable lipid at a level of about 6% of the total fatty acidcontent.
 99. The process of claim 86, wherein DHA is present in theextractable lipid at a level of between 7% and 20% of the total fattyacid content.